U.S. patent application number 15/016375 was filed with the patent office on 2016-09-01 for vector co-expressing vaccine and costimulatory molecules.
The applicant listed for this patent is HEAT BIOLOGICS, INC.. Invention is credited to George Fromm, Taylor Schreiber.
Application Number | 20160250322 15/016375 |
Document ID | / |
Family ID | 56564722 |
Filed Date | 2016-09-01 |
United States Patent
Application |
20160250322 |
Kind Code |
A1 |
Schreiber; Taylor ; et
al. |
September 1, 2016 |
VECTOR CO-EXPRESSING VACCINE AND COSTIMULATORY MOLECULES
Abstract
Compositions and methods for co-expressing a secretable vaccine
protein (such as gp96-Ig) and T-cell co-stimulatory molecules from
a single vector, among others, are provided herein. Materials and
methods for using gp96-Ig vaccination and T-cell co-stimulation to
treat a clinical condition (e.g., cancer) in a subject also are
provided.
Inventors: |
Schreiber; Taylor; (DURHAM,
NC) ; Fromm; George; (DURHAM, NC) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
HEAT BIOLOGICS, INC. |
DURHAM |
NC |
US |
|
|
Family ID: |
56564722 |
Appl. No.: |
15/016375 |
Filed: |
February 5, 2016 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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62174942 |
Jun 12, 2015 |
|
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62113153 |
Feb 6, 2015 |
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Current U.S.
Class: |
424/172.1 |
Current CPC
Class: |
A61P 31/20 20180101;
C07K 2319/40 20130101; A61K 2039/505 20130101; A61P 35/00 20180101;
A61K 2039/53 20130101; C07K 14/4702 20130101; A61K 39/42 20130101;
A61K 2039/5258 20130101; A61K 39/39 20130101; A61K 2039/5152
20130101; A61K 2039/57 20130101; A61P 31/12 20180101; C07K 2317/75
20130101; A61K 39/3955 20130101; C07K 14/70532 20130101; C07K
16/2878 20130101; A61P 31/14 20180101; C07K 2319/30 20130101; A61K
2039/55516 20130101; A61P 31/18 20180101; A61K 39/0011 20130101;
C12N 15/86 20130101; Y02A 50/30 20180101; A61P 33/02 20180101; A61P
43/00 20180101; C07K 14/70575 20130101; C07K 2319/75 20130101; A61K
2039/525 20130101; A61K 2039/5156 20130101; A61K 48/00 20130101;
A61K 39/0011 20130101; A61K 2300/00 20130101 |
International
Class: |
A61K 39/39 20060101
A61K039/39; C07K 14/705 20060101 C07K014/705; C07K 14/47 20060101
C07K014/47; A61K 39/395 20060101 A61K039/395; A61K 39/00 20060101
A61K039/00 |
Claims
1. An expression vector comprising a first nucleotide sequence that
encodes a secretable vaccine protein, and a second nucleotide
sequence that encodes a T cell costimulatory fusion protein,
wherein the T cell costimulatory fusion protein enhances activation
of antigen-specific T cells when administered to a subject.
2. The expression vector of claim 1, wherein the vector is a
mammalian expression vector.
3. The expression vector of claim 1, wherein the vaccine protein is
a secretable 96-Ig fusion protein which optionally lacks the gp96
KDEL (SEQ ID NO:3) sequence.
4. The expression vector of claim 3, wherein the Ig tag in the
gp96-Ig fusion protein comprises the Fc region of human IgG1, IgG2,
IgG3, IgG4, IgM, IgA, or IgE.
5. The expression vector of claim 1, wherein the T cell
costimulatory fusion protein is OX40L-Ig, or a portion thereof that
binds to OX40.
6. The expression vector of claim 1, wherein the T cell
costimulatory fusion protein is ICOSL-Ig, or a portion thereof that
binds to ICOS.
7. The expression vector of claim 1, wherein the T cell
costimulatory fusion protein is 4-1BBL-Ig, or a portion thereof
that binds to 4-1BBR.
8. The expression vector of claim 1, wherein the T cell
costimulatory fusion protein is TL1A-Ig, or a portion thereof that
binds to TNFRSF25.
9. The expression vector of claim 1, wherein the T cell
costimulatory fusion protein is GITRL-Ig, or a portion thereof that
binds to GITR.
10. The expression vector of claim 1, wherein the T cell
costimulatory fusion protein is CD40L-Ig, or a portion thereof that
binds to CD40.
11. The expression vector of claim 1, wherein the T cell
costimulatory fusion protein is CD70-Ig, or a portion thereof that
binds to CD27.
12. The expression vector of any one of claims 5-11, wherein the Ig
tag in the T cell costimulatory fusion protein comprises the Fc
region of human IgG1, IgG2, IgG3, IgG4, IgM, IgA, or IgE.
13. The expression vector of claim 1, wherein the expression vector
comprises DNA.
14. The expression vector of claim 1, wherein the expression vector
comprises RNA.
15. A composition comprising an expression vector that comprises a
first nucleotide sequence encoding a secretable vaccine protein,
and a second nucleotide sequence encoding a T cell costimulatory
fusion protein, wherein the T cell costimulatory fusion protein
enhances activation of antigen-specific T cells when administered
to a subject.
16. The composition of claim 15, wherein the vector is a DNA-based
mammalian expression vector.
17. The composition of claim 15, wherein the secretable vaccine
protein is a secretable gp96-Ig fusion protein which optionally
lacks the gp96 KDEL (SEQ ID NO:3) sequence.
18. The composition of claim 17, wherein the Ig tag in the gp96-Ig
fusion protein comprises the Fc region of human IgG1, IgG2, IgG3,
IgG4, IgM, IgA, or IgE.
19. The composition of claim 15, wherein the T cell costimulatory
fusion protein is OX40L-Ig, or a portion thereof that binds to
OX40.
20. The composition of claim 15, wherein the T cell costimulatory
fusion protein is ICOSL-Ig, or a portion thereof that binds to
ICOS.
21. The composition of claim 15, wherein the T cell costimulatory
fusion protein is 4-1BBL-Ig, or a portion thereof that binds to
4-1BBR.
22. The composition of claim 15, wherein the T cell costimulatory
fusion protein is TL1A-Ig, or a portion thereof that binds to
TNFRSF25.
23. The composition of claim 15, wherein the T cell costimulatory
fusion protein is GITRL-Ig, or a portion thereof that binds to
GITR.
24. The composition of claim 15, wherein the T cell costimulatory
fusion protein is CD40L-Ig, or a portion thereof that binds to
CD40.
25. The composition of claim 15, wherein the T cell costimulatory
fusion protein is CD70-Ig, or a portion thereof that binds to
CD27.
26. The composition of any one of claims 19-25, wherein the Ig tag
in the T cell costimulatory fusion protein comprises the Fc region
of human IgG1, IgG2, IgG3, IgG4, IgM, IgA, or IgE.
27. The composition of any one of claims 15-26, wherein the
expression vector is incorporated into a virus or virus-like
particle.
28. The composition of any one of claims 15-26, wherein the
expression vector is incorporated into a human tumor cell.
29. The composition of claim 28, wherein the human tumor cell is a
cell from an established NSCLC, bladder cancer, melanoma, ovarian
cancer, renal cell carcinoma, prostate carcinoma, sarcoma, breast
carcinoma, squamous cell carcinoma, head and neck carcinoma,
hepatocellular carcinoma, pancreatic carcinoma, or colon carcinoma
cell line.
30. A method for treating a subject, comprising administering to a
subject an effective amount of a composition comprising an
expression vector that comprises a first nucleotide sequence
encoding a secretable vaccine protein, and a second nucleotide
sequence encoding a T cell costimulatory fusion protein, wherein
the T cell costimulatory fusion protein enhances activation of
antigen-specific T cells when administered to the subject.
31. The method of claim 30, wherein the vector is incorporated into
a virus or virus-like particle.
32. The method of claim 30, wherein the vector is incorporated into
a human tumor cell.
33. The method of any one of claims 30-32, wherein the subject is a
human cancer patient.
34. The method of claim 33, wherein administration of the
composition to the human patient increases the activation or
proliferation of tumor antigen specific T cells in the patient.
35. The method of claim 34, wherein the activation or proliferation
of tumor antigen specific T cells in the patient is increased by at
least 25 percent as compared to the level of activation or
proliferation of tumor antigen specific T cells in the patient
prior to the administration.
36. The method of claim 30, comprising administering the
composition to a human cancer patient in combination with an agent
that inhibits immunosuppressive molecules produced by tumor
cells.
37. The method of claim 36, wherein the agent is an antibody
against PD-1.
38. The method of any one of claims 30-37, wherein the subject is a
human with an acute or chronic infection.
39. The method of claim 38, wherein the acute or chronic infection
is an infection by hepatitis C virus, hepatitis B virus, human
immunodeficiency virus, or malaria.
40. The method of claim 38, wherein administration of the
composition to the human patient stimulates the activation or
proliferation of pathogenic antigen specific T cells.
41. The method of claim 30, wherein the T cell costimulatory
molecule enhances the activation of antigen-specific T cells in the
subject to a greater level than gp96-Ig vaccination alone.
42. The method of any one of claims 30-41, wherein the secretable
vaccine protein is a secretable 96-Ig fusion protein.
Description
RELATED APPLICATIONS
[0001] The present application claims priority to U.S. Provisional
Patent Application Nos. 62/113,153, filed Feb. 6, 2015, and
62/174,942, filed Jun. 12, 2015, the entire contents of all of
which are hereby incorporated by reference.
TECHNICAL FIELD
[0002] This document relates, inter alia, to materials and methods
for using vaccination and T-cell co-stimulation to treat a clinical
condition in a subject, including materials and methods for
co-expressing a vaccine (e.g. gp96-Ig) and T-cell co-stimulatory
molecules from a single vector.
Description of the Text File Submitted Electronically
[0003] The contents of the text file submitted electronically
herewith are incorporated herein by reference in their entirety: A
computer readable format copy of the Sequence Listing (filename
HTB-021-SequenceListing.txt; date recorded: Feb. 4, 2016; file
size: 73 KB).
BACKGROUND
[0004] Cancer is a disease that arises from a prolonged period of
genetic instability that extends the lifespan of a normal cell. The
triggering event that marks the beginning of this period is
variable between cell types, but commonly is the acquisition of a
mutation in a tumor suppressor gene such as p53 or Rb, a mutation
in a proto-oncogene such as KRAS or myc, or infection of a cell
with an oncogenic virus such as HPV16 or EBV. Whatever the origin,
cells that acquire mutations in genes that enable them to escape
normal growth controls or cell death pathways become more likely to
acquire additional mutations. Once a cell has acquired "enough"
mutations, typically thought to be at least six, it no longer is
responsive to intrinsic or extrinsic signals that would restrain
its growth or trigger apoptosis.
[0005] Because tumors arise from host cells, the body's immune
system is initially tolerant to those cells. The acquisition of
tumorigenic mutations may or may not lead to production of a
mutated protein containing an epitope that is sufficiently non-self
to become immunogenic. If a cell acquires an immunogenic mutation,
it can be sought out and destroyed by the host immune system, a
process known as immunosurveillance (Smyth et al., Adv Immunol
2006, 90:1-50). Murine studies have provided support for the immune
surveillance hypothesis (Dunn et al., Nat Immunol 2002, 3:991-998;
Shankaran et al., Nature 2001, 410:1107-1111; and Dunn et al., Annu
Rev Immunol 2004, 22:329-360), and also suggested that innate in
addition to so-called adaptive immune responses may facilitate
rejection of immunogenic tumors (Unni et al., Proc Natl Acad Sci
USA 2008, 105:1686-1691; Taieb et al., Nat Med 2006, 12:214-219;
and Raulet and Guerra, Nat Rev Immunol 2009, 9:568-580). Innate
responses can be evoked through induced expression of NK activating
signals such as NKG2D ligand expression or following DNA damage
incurred as a result of mutagenic or viral processes. Some cells
that acquire immunogenic mutations also gain the capacity to engage
normal immune regulatory systems that dampen anti-self immune
responses (Rabinovich et al., Annu Rev Immunol 2007, 25:267-296).
The pathways driving the activation of host regulatory mechanisms
are poorly understood. Still other cells may gain a number of
oncogenic mutations without ever producing an immunogenic peptide
that leads to activation of the host immune system. Therefore,
tumor cells that produce an immunogenic peptide during their
transformation must continuously evade anti-tumor immune responses
in order to survive, whereas tumors that become transformed without
activating the immune system may not rely on such immune regulatory
mechanisms for survival.
SUMMARY
[0006] It is possible that combination therapies including
combinations or subcombinations of one or more checkpoint
inhibitors, one or more vaccines, and one or more T cell
costimulatory molecules may expand the base of cancer patients that
can benefit from immunotherapy. Vaccines may contribute to this
response by increasing both the frequency of tumor-antigen specific
CD8+ T cells and also the number of tumor antigens recognized by
those CD8+ T cells. T cell costimulatory molecules may enhance the
response by further increasing the frequency and/or enhancing the
activation of tumor antigen-specific T cells, and also by
increasing the expression of tumor-killing effector molecules by
CD8+ T cells. When used in combination with checkpoint inhibitors,
it may be possible to generate a broad range of highly activated
CD8+ T cells that will be able to infiltrate tumors and will not be
inhibited by various checkpoint pathways once infiltration has
occurred. An impediment to the success of combination therapies,
however, is that they traditionally require administration of at
least three different drug products (a vaccine, a T cell
costimulatory, and a checkpoint inhibitor), each of which has a
significant cost and, in some cases, toxicity.
[0007] This document is based, at least in part, on the discovery
that a combination of a vaccination, e.g. gp96-Ig vaccination, and
T cell costimulation with one or more agonists of OX40, ICOS,
4-1BB, TNFRSF25, CD40, CD27, and/or GITR, among others, provides a
synergistic anti-tumor benefit. Pre-clinical models have evaluated
independent compositions of 96-Ig vaccines combined with agonistic
antibodies targeting OX40, ICOS, 4-1BB, and TNFRSF25, and
demonstrated variable effects on mechanistic and anti-tumor
complementarity. The materials and methods described herein are
advantageous in that, inter alia, they provide a single composition
that can achieve both vaccination with, for example, gp96-Ig, and T
cell costimulation without the need for independent products. These
materials and methods achieve this goal by creating a single
vaccine protein (e.g., gp96-Ig) expression vector that has been
genetically modified to simultaneously express an costimulatory
molecule, including without limitation, fusion proteins such as
ICOSL-Ig, 4-1BBL-Ig, TL1A-Ig, OX40L-Ig, CD40L-Ig, CD70-Ig, or
GITRL-Ig to provide T cell costimulation. The vectors, and methods
for their use, can provide a costimulatory benefit without the need
for an additional antibody therapy to enhance the activation of
antigen-specific CD8+ T cells. Thus, combination immunotherapy can
be achieved by vector re-engineering to obviate the need for
vaccine/antibody/fusion protein regimens, which may reduce both the
cost of therapy and the risk of systemic toxicity.
[0008] In one aspect, this document features an expression vector
containing a first nucleotide sequence that encodes a secretable
vaccine protein, and a second nucleotide sequence that encodes a T
cell costimulatory fusion protein, wherein the T cell costimulatory
fusion protein enhances activation of antigen-specific T cells when
administered to a subject. In some embodiments, this document
features an expression vector containing a first nucleotide
sequence that encodes a secretable gp96-Ig fusion protein, and a
second nucleotide sequence that encodes a T cell costimulatory
fusion protein, wherein the T cell costimulatory fusion protein
enhances activation of antigen-specific T cells when administered
to a subject. The expression vector can be a mammalian expression
vector. In an embodiment, the secretable gp96-Ig fusion protein can
lack the gp96 KDEL (SEQ ID NO:3) sequence. The Ig tag in the
gp96-Ig fusion protein can include the Fc region of human IgG1,
IgG2, IgG3, IgG4, IgM, IgA, or IgE. The T cell costimulatory fusion
protein can be OX40L-Ig or a portion thereof that binds to OX40,
ICOSL-Ig or a portion thereof that binds to ICOS, 4-1BBL-Ig or a
portion thereof that binds to 4-1BBR, TL1A-Ig or a portion thereof
that binds to TNFRSF25, GITRL-Ig or a portion thereof that binds to
GITR, CD40-Ig or a portion thereof that binds to CD40, or CD70-Ig
or a portion thereof that binds to CD27, among others. The Ig tag
in the T cell costimulatory fusion protein can include the Fc
region of human IgG1, IgG2, IgG3, IgG4, IgM, IgA, or IgE. The
expression vector can contain DNA or RNA.
[0009] In another aspect, this document features a composition
containing an expression vector that comprises a first nucleotide
sequence encoding a secretable vaccine protein, such as a
secretable gp96-Ig fusion protein, and a second nucleotide sequence
encoding a T cell costimulatory fusion protein, wherein the T cell
costimulatory fusion protein enhances activation of
antigen-specific T cells when administered to a subject. The vector
can be a DNA-based mammalian expression vector. In an embodiment,
the secretable gp96-Ig fusion protein can lack the gp96 KDEL (SEQ
ID NO:3) sequence. The Ig tag in the gp96-Ig fusion protein can
contain the Fc region of human IgG1, IgG2, IgG3, IgG4, IgM, IgA, or
IgE. The T cell costimulatory fusion protein can be OX40L-Ig or a
portion thereof that binds to OX40, ICOSL-Ig or a portion thereof
that binds to ICOS, 4-1BBL-Ig or a portion thereof that binds to
4-1BBR, TL1A-Ig or a portion thereof that binds to TNFRSF25,
GITRL-Ig or a portion thereof that binds to GITR, CD40L-Ig or a
portion thereof that binds to CD40, or CD70-Ig or a portion thereof
that binds to CD27. The Ig tag in the T cell costimulatory fusion
protein can include the Fc region of human IgG1, IgG2, IgG3, IgG4,
IgM, IgA, or IgE. The expression vector can be incorporated into a
virus or virus-like particle, or can be incorporated into a human
tumor cell (e.g., a human tumor cell from an established cell line,
e.g. a NSCLC, bladder cancer, melanoma, ovarian cancer, renal cell
carcinoma, prostate carcinoma, sarcoma, breast carcinoma, squamous
cell carcinoma, head and neck carcinoma, hepatocellular carcinoma,
pancreatic carcinoma, or colon carcinoma cell line).
[0010] In another aspect, this document features a cell comprising
a composition containing an expression vector that comprises a
first nucleotide sequence encoding a secretable vaccine protein,
and a second nucleotide sequence encoding a T cell costimulatory
fusion protein, wherein the T cell costimulatory fusion protein
enhances activation of antigen-specific T cells when administered
to a subject. In some embodiments, this document features a cell
comprising a composition containing an expression vector that
comprises a first nucleotide sequence encoding a secretable gp96-Ig
fusion protein, and a second nucleotide sequence encoding a T cell
costimulatory fusion protein, wherein the T cell costimulatory
fusion protein enhances activation of antigen-specific T cells when
administered to a subject. Such a cell, in various embodiments, can
be suitable for use as an off-the-shelf therapy. Such a cell, in
various embodiments, is irradiated. Such a cell, in various
embodiments, is live and attenuated. These cells, in various
embodiments, express tumor antigens which may be chaperoned by the
vaccine protein (e.g., gp96) of the present compositions. Such a
cell, in various embodiments, can be derived from an established
cell line e.g., a human tumor cell from an established NSCLC,
bladder cancer, melanoma, ovarian cancer, renal cell carcinoma,
prostate carcinoma, sarcoma, breast carcinoma, squamous cell
carcinoma, head and neck carcinoma, hepatocellular carcinoma,
pancreatic carcinoma, or colon carcinoma cell line. Such a cell, in
various embodiments, can be derived from an established prostate
cancer cell line. Such a cell, in various embodiments, can be
derived from an established lung cancer cell line. Such a cell, in
various embodiments, can be derived from an established bladder
cancer cell line. Such a cell, in various embodiments, can be
derived from an established sarcoma cell line. Such a cell, in
various embodiments, can be derived from an established
choriocarcinoma cancer cell line.
[0011] In another aspect, this document features a method for
treating a subject. The method can include administering to a
subject an effective amount of a composition described herein, for
instance, containing an expression vector that includes a first
nucleotide sequence encoding a secretable vaccine protein such as a
secretable gp96-Ig fusion protein, and a second nucleotide sequence
encoding a T cell costimulatory fusion protein, wherein the T cell
costimulatory fusion protein enhances activation of
antigen-specific T cells when administered to the subject. The
vector can be incorporated into a virus or virus-like particle, or
incorporated into a human tumor cell. The subject can be a human
cancer patient. Administration of the composition to the human
patient can increase the activation or proliferation of tumor
antigen specific T cells in the patient. For example, the
activation or proliferation of tumor antigen specific T cells in
the patient can be increased by at least 25 percent (e.g., at least
30 percent, at least 40 percent, at least 50 percent, at least 60
percent, at least 70 percent, or at least 75 percent) as compared
to the level of activation or proliferation of tumor antigen
specific T cells in the patient prior to the administration. The
method can include administering the composition to a human cancer
patient in combination with an agent that inhibits
immunosuppressive molecules produced by tumor cells. The agent can
be an antibody against PD-1. The subject can be a human with an
acute or chronic infection (e.g., an infection by hepatitis C
virus, hepatitis B virus, human immunodeficiency virus, or
malaria). Administration of the composition to the human patient
can stimulate the activation or proliferation of pathogenic antigen
specific T cells. The T cell costimulatory molecule can enhance the
activation of antigen-specific T cells in the subject to a greater
level than gp96-Ig vaccination alone.
[0012] Unless otherwise defined, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention pertains.
Although methods and materials similar or equivalent to those
described herein can be used to practice the invention, suitable
methods and materials are described below. All publications, patent
applications, patents, and other references mentioned herein are
incorporated by reference in their entirety. In case of conflict,
the present specification, including definitions, will control. In
addition, the materials, methods, and examples are illustrative
only and not intended to be limiting.
[0013] The details of one or more embodiments of the invention are
set forth in the accompanying drawings and the description below.
Other features, objects, and advantages of the invention will be
apparent from the description and drawings, and from the
claims.
DESCRIPTION OF DRAWINGS
[0014] FIG. 1 is a schematic representation of the re-engineering
of an original gp96-Ig vector to generate a cell-based combination
product that encodes the gp96-Ig fusion protein in a first
cassette, and a T cell costimulatory fusion protein in a second
cassette. ICOS-Fc, 4-1BBL-Fc, and OX40L-Fc are shown for
illustration.
[0015] FIG. 2 is a schematic representation of a mammalian
expression vector (B45) encoding a secretable gp96-Ig fusion
protein in one expression cassette and a T cell costimulatory
fusion protein (by way of non-limiting illustration, ICOSL-IgG4 Fc)
in a second cassette.
[0016] FIG. 3 is an illustration of an allogeneic tumor cell that
has been transfected with a vector encoding two secretable
proteins. The first protein, gp96-Ig, forms a secretable dimer
(smooth) that chaperones cell-derived antigens outside the cells.
The second protein is a trimeric secretable T cell costimulatory
fusion protein (rough) which is secreted by the vaccine cell and
may freely bind to a nearby costimulatory receptor on the surface
of a T cell.
[0017] FIGS. 4A-4G show that an OX40 agonist antibody in
combination with 96-Ig cellular vaccine promotes antigen specific
CD8 proliferation, while FOXP3+ Tregs remain unaffected. FIGS.
4A-4D depict the 96-Ig cellular vaccine mechanism of action. In
FIG. 4A, vaccine cells secrete gp96-Ig along with cell-derived
antigens, or in the case of FIGS. 5, 6, and 8, the single antigen
chicken ovalbumin which is stably expressed in this vaccine cell
line. In FIG. 4B, gp96-Ig/antigen complexes are taken up by APCs
and the antigens are transferred to MHC class I molecules. In FIG.
4C, antigen cross-presentation leads to CD8+ specific T cell
activation. In FIG. 4D, in the context of a tumor, the activated
CD8+ T cells can recognize shared tumor antigens on distant tumors
and destroy them. FIG. 4E is a graph plotting antigen specific
(OT-1) cell expansion following vaccination with ImPACT (as used
herein, this refers to a modified (e.g. KDEL deletion) gp96-Ig
fusion protein or, in some cases, an engineered cell line designed
to express the gp96-Ig fusion protein) alone or in combination with
agonistic T cell co-stimulatory antibodies for ICOS, 4-1BB, or
OX40. Data are shown for days 5 and 40 following the initial
vaccination (prime and memory, respectively). The latter also
corresponds to 5 days after the second, boost vaccination. Only the
ImPACT/OX40(ab) combination generated OT-1 expansion that was
significantly greater than ImPACT on its own (*, p<0.05).
Experimental replicates are listed. Plotted values are the mean and
error is SEM. For each data set in FIG. 4E, the order of histograms
from left to right is: No Vaccine, ImPACT alone, ImPACT plus ICOS
antibody, ImPact plus OX40 antibody, and ImPact plus 4-1BB
antibody. FIG. 4F is a graph plotting FOXP3+ Tregs as a percentage
of total CD4+ cells. With the exception of ImPACT/4-1BB treated
mice, which fluctuated from increased to decreased FOXP3+ cells,
there was no significant change in Tregs following ImPACT
treatment, emphasizing its specificity towards CD8+ expansion.
Experimental replicates are listed. Plotted values are the mean and
error is SEM. For each data set in FIG. 4F, the order of histograms
from left to right is: No Vaccine, ImPACT alone, ImPact plus ICOS
antibody, ImPact plus OX40 antibody, and ImPact plus 4-1BB
antibody. FIG. 4G is a pair of graphs plotting OT-1/CD8 and FOXP3+
cell expansion in a second model system in response to the adjuvant
Alum. Again, the combination of ImPACT and OX40(ab) resulted in
significant OT-1 proliferation, in addition to a moderate increase
in FOXP3+ cells. Experimental replicates are listed. Plotted values
are the mean and error is SEM. For each data set in FIG. 4G, the
order of histograms from left to right is: No Vaccine, IgG control,
and ImPact plus OX40 antibody.
[0018] FIGS. 5A-5D show that T cell co-stimulator OX40 functions
synergistically with the ImPACT (gp96-Ig) cellular vaccine to
activate T cells and produce antigen specific CD8+ expansion. FIG.
5A is a schematic representation of receptor (OX40, ICOS, and
4-1BB) and ligand (OX40L, ICOSL, and 4-1BBL) interactions between T
cells and antigen presenting cells (APC) promoting T cell
activation. FIG. 5B is a diagram depicting a vaccine cell line
established through selection of a clonal population of cells
expressing gp96-Ig/HLA-A1 (ImPACT) along with the single antigen
chicken ovalbumin, in order to track antigen specific T cell
expansion (OT-I cells) following the administration of vaccine.
FIG. 5C is a list of murine T cell co-stimulator agonist antibodies
tested in combination with ImPACT for synergism in promoting T cell
expansion--all of these antibodies are useful in various
embodiments as combination therapy agents. FIG. 5D is a graph
plotting OT-1 levels in FOXP3-RFP reporter mice that were seeded
with antigen specific OT-1 (CD8) cells labeled with GFP via tail
vein injection on day -1, as detected by flow cytometry for 43 days
after vaccination with either ImPACT alone, or ImPACT in
combination with 100 .mu.g of agonist T cell co-stimulatory
antibodies for OX40, ICOS, or 4-1BB on day 0. Unvaccinated (No
Vaccine) mice were assessed in parallel as a control. Mice were
boosted with vaccine or vaccine/antibody combinations again on day
35. Vaccination days are indicated by syringes. The initial (prime)
response peaked on day 5, and only vaccine/OX40(ab) treated mice
showed a modest memory response following boost (arrows). Plotted
values represent the mean percentage of OT-1 cells from all CD8+
cells and error is SEM. See also FIGS. 4A-G for the number of
experimental replicates and statistical significance between sample
groups.
[0019] FIGS. 6A-6C show that the combination of T cell
co-stimulator OX40L with ImPACT into a new vaccine vector
("ComPACT") produced surprisingly superior antigen specific CD8+ T
cell expansion as compared to coadministration of OX40 agonist
antibody. FIG. 6A depicts the experimental design to compare (a)
antigen specific T cell expansion using the original vaccine ImPACT
(Gp96-Ig) in combination with OX40 agonist antibody to (b) the new
vaccine ComPACT (in this figure, Gp96-Ig/OX40L-Fc). FIG. 6B depicts
the peak of antigen-specific CD8+ T cell proliferation following
primary immunization with a control vaccine, a vaccine expressing
ovalbumin, a vaccine expressing ovalbumin and gp96-Ig (ImPACT),
ImPACT in combination with OX40 agonist antibodies, or ComPACT. For
each data set in FIG. 6B, the order of peaks from left to right is:
No Vaccine, Ova only control, ImPACT, ImPACT plus OX40 antibody,
and ComPACT. FIG. 6C is a graph plotting the OT-1 expansion
time-course (similar to FIG. 5D), using FOXP3-RFP mice seeded with
OT-1 (CD8) cells on day -1. OT-1/GFP cells were analyzed by flow
cytometry in mice treated with No Vaccine, Ova only control cells,
ImPACT, ImPACT+100 .mu.g OX40(ab), and ComPACT, over 46 days, with
initial vaccination on day 0 and a boost on day 35 (indicated by
syringes). Both prime and memory responses (arrows) were greatest
in mice treated with ComPACT, even when compared with
ImPACT+OX40(ab). ComPACT mice also surprisingly retained elevated
OT-1 levels throughout the time-course (.about.days 7-20). Plotted
values represent the mean and error is SEM. See also FIGS. 7A-7F
for the number of experimental replicates and statistical
significance between sample groups.
[0020] FIGS. 7A-7F show that the combination of gp96-Ig and OX40L,
ICOSL, or 4-1BBL expression in ComPACT results in high-level CD8,
antigen specific T cell response. FIGS. 7A-7D show the
characterization of the 3T3-version of ComPACT as used in FIG. 6C.
3T3 cells were transfected with a plasmid expressing chicken
ovalbumin (Ova), and a single high-expressing clone was established
and used to re-transfect with Vaccine vectors (either gp96-Ig
alone, gp96-Ig/OX40L-Fc, gp96-Ig/ICOSL, or gp96-Ig/4-1BBL).
Vaccines were therefore established in the same Ova parental clone.
Unvaccinated mice (No vaccine) were compared with mice treated with
Ova only expressing cells (as an additional control), ImPACT
(Ova-gp96-Ig), ImPACT+OX40 agonist antibody (OX40(ab)), ComPACT
(Ova-gp96-Ig/ICOSL), ComPACT (Ova-gp96-Ig/OX40L-Fc), or ComPACT
(Ova-gp96-Ig/1BBL). FIG. 7A is a graph plotting Ova secretion as
confirmed by ELISA, showing that secretion was essentially
identical between Ova only control, ImPACT, and the various ComPACT
cells. Values are the mean from a minimum of 6 replicates and error
is SEM. For each data set in FIG. 7A, the order of histograms from
left to right is: Ova only control, ImPACT (3T3-ova-gp96-Ig),
ComPACT (Ova-gp96-Ig/ICOSL), ComPACT (Ova-gp96-Ig/OX40L-Fc), and
ComPACT (Ova-gp96-Ig/1BBL). FIG. 7B is a graph plotting gp96-Ig
secretion (detected as IgG) as determined by ELISA, showing that
individual ImPACT and ComPACT clones were established that secreted
comparable levels. Values are the mean from a minimum of 6
replicates and error is SEM. For each data set in FIG. 7B, the
order of histograms from left to right is: Ova only control, ImPACT
(3T3-ova-gp96-Ig), ComPACT (Ova-gp96-Ig/ICOSL), ComPACT
(Ova-gp96-Ig/OX40L-Fc), and ComPACT (Ova-gp96-Ig/1BBL). FIG. 7C is
a graph plotting mRNA expression of ICOSL, OX40L, or 4-1BBL as
confirmed by qRT-PCR, showing expression only in ComPACT cells.
Graphical values are the mean from a minimum of 3 distinct
replicates and error is SEM. For each data set in FIG. 7C, the
order of histograms from left to right is: Ova only control, ImPACT
(3T3-ova-gp96-Ig), ComPACT (Ova-gp96-Ig/ICOSL), ComPACT
(Ova-gp96-Ig/OX40L-Fc), and ComPACT (Ova-gp96-Ig/1BBL). FIG. 7D
provides Western blots showing confirmation of OX40L, ICOSL, and
4-1BBL expression in ComPACT cells. ImPACT and ComPACT cells were
treated with Brefeldin A (BFA) for 16 hours to prevent protein
transport and secretion. Cells were then harvested, lysed and
subjected to SDS PAGE/Western blot analysis. Blots were probed with
an antibody to OX40L (also known as CD252), ICOSL, or 4-1BBL, and
histone H3 or actin B (ACTB) as a loading control. OX40L, ICOSL,
and 4-1BBL were only detected in ComPACT cells. FIG. 7E is a graph
plotting the frequency of CD4+FoxP3+ regulatory T cells on day 5
following the indicated primary immunization. For each data set in
FIG. 7E, the order of peaks from left to right is: No vaccine, Ova
only control, ImPACT, ImPACT plus OX40 antibody, and ComPACT (in
this figure, Ova-gp96-Ig/OX40L-Fc). FIG. 7F is a pair of graphs
plotting the frequency of antigen-specific CD8+ T cells (OT-I) in
the peripheral blood on day 42 (7 days following the boost
immunization), shown on the left, and the peak of CD4+FoxP3+ T
regulatory cells on the same day in the peripheral blood. As in
FIG. 6C, antigen specific (OT-1) cell expansion following
vaccination with an Ova only expressing cell line, ImPACT, ImPACT
in combination with OX40(ab) and ComPACT (in this case,
Ova-gp96-Ig/OX40L-Fc), are shown at 5 and 40 days following the
initial vaccination (prime and memory, respectively). The latter
also corresponds to 5 days after the second, boost vaccination.
OT-1 levels in ImPACT, ImPACT+OX40(ab) and ComPACT treated mice are
significantly elevated compared to Ova only control treated mice.
ComPACT treated mice exhibit the greatest proliferation of OT-1
cells, which are significantly higher than ImPACT+OX40(ab) at both
prime and memory response points. Experimental replicates are
listed and error is SEM. For each data set in FIG. 7F, the order of
peaks from left to right is: No vaccine, Ova only control, ImPACT,
ImPACT plus OX40 antibody, and ComPACT (in this figure,
Ova-gp96-Ig/OX40L-Fc).
[0021] FIGS. 8A-8E show that ComPACT elicited antigen specific CD8+
expansion while OX40 antibody led to non-specific T cell
activation. FIG. 8A is a series of graphs plotting total numbers of
mononuclear (MNC), CD4, CD8, OT-I, and OT-II cells in mice that
were either untreated or vaccinated with ImPACT, ImPACT+OX40(ab),
or ComPACT (in this figure, Gp96-Ig/OX40L-Fc). As for FIGS. 5D and
6C, FOXP3-RFP reporter mice were seeded with OT-1 cells via tail
vein injection on day -1, vaccinated on day 0, and sacrificed on
day 8 for analysis, including flow cytometry of cells obtained from
peritoneal wash. ComPACT treatment produced a robust OT-I (CD8)
specific response, whereas OX40(ab) treatment resulted in an
increase in all T cell sub-types, including FOXP3+ CD4 cells.
Plotted values represent the mean from a minimum of 3 mice and
error is SEM. For each data set in FIG. 8A, the order of peaks from
left to right is: Untreated, ImPACT, ImPACT plus OX40 antibody, and
ComPACT (in this figure, Gp96-Ig/OX40L-Fc). FIG. 8B is a series of
graphs plotting numbers of CD127.sup.+KLRG1.sup.-,
CD127.sup.-KLRG1.sup.+, and CD127.sup.+KLRG1.sup.+ cells, which
correspond to memory precursor cells, short-lived effector cells,
and memory cells, respectively, on day 8 following the primary
immunization. The cells were derived from the spleen (top panels)
and peritoneal cavity (bottom panels). For each data set in FIG.
8B, the order of peaks from left to right is: Untreated, Ova only,
ImPACT, ComPACT (Ova-gp96-Ig/ICOSL), ComPACT
(Ova-gp96-Ig/OX40L-Fc), and ComPACT (Ova-gp96-Ig/1BBL). FIG. 8C is
a series of graphs plotting levels of INF.gamma., TNF.alpha., IL2,
IL6, and IL5. Whole blood serum was harvested from the same mice
presented in FIG. 8A above on day 8, and subjected to cytokine
analysis using the LEGENDPLEX.TM. kit from BioLegend and flow
cytometer. Consistent with the data of FIG. 8A, OX40(ab) treatment
produced a non-specific, systemic immune response, with elevated
levels of not only INF.gamma., TNF.alpha. and IL2, but also IL6 and
IL5. Plotted values represent the mean from a minimum of 3 mice and
error is SEM. For each data set in FIG. 8C, the order of peaks from
left to right is: Untreated, ImPACT, ImPACT plus OX40 antibody, and
ComPACT (in this figure, Gp96-Ig/OX40L-Fc). FIG. 8D is a series of
graphs plotting gene expression levels for FN.gamma.TNF.alpha., and
Granzyme-B (GZMB). Analysis of T cell activation genes by qRT-PCR
demonstrated ComPACT's specificity in only activating antigen
specific CD8 (OT-I+) cells, compared to OX40(ab), which
non-specifically activated both endogenous (OT-I-) and antigen
specific CD8 (OT-I+) cells. Cells obtained from peritoneal washes
in FIG. 8A above were sorted into populations of OT-1- and OT-1+
CD8 cells. Total RNA was harvested, reverse transcribed and
analyzed by qPCR. Gene expression levels of IFN.gamma., TNF.alpha.,
and GZMB are shown, normalized to 18S mRNA with the first ImPACT
only treated replicate set at 1. Plotted values represent the mean
from a minimum of 3 mice, and error is SEM. For each data set in
FIG. 8D, the order of histograms from left to right is: ImPACT,
ImPACT plus OX40 antibody, and ComPACT (in this figure,
Gp96-Ig/OX40L-Fc). FIG. 8E shows the number of FOXP3 regulatory T
cells (Treg) in splenocytes and tumor draining lymph node (TDLN) in
the mice. For each data set in FIG. 8E, the order of peaks from
left to right is: Untreated, ImPACT, ImPACT plus OX40 antibody, and
ComPACT (in this figure, Gp96-Ig/OX40L-Fc).
[0022] FIGS. 9A-9C show that ComPACT (in this figure,
Gp96-Ig/OX40L-Fc) treatment results in antigen specific CD8 T cell
activation, whereas coadministration of OX40(ab) treatment elicits
non-specific immune cell activation including increases in FOXP3
Tregs in both the spleen and lymph nodes. FIG. 9A is a series of
graphs plotting total numbers of MNC, CD4, CD8, OT-I, and OT-II
cells for mice either untreated or vaccinated with ImPACT,
ImPACT+OX40(ab), or ComPACT. As in FIGS. 5D and 6C, FIR reporter
mice were seeded with OT-1 cells via tail vein injection on day -1,
vaccinated on day 0, and sacrificed on day 8 for analysis,
including flow cytometry of cells obtained from the spleen.
OX40(ab) treated mice demonstrated an increase in all T cell
sub-types, including CD4/FOXP3+ cells. ComPACT treated mice
produced a robust OT-1 (CD8) specific response that was
significantly higher than the OX40(ab) response. Plotted values
represent the mean from a minimum of 3 mice and error is SEM. For
each data set in FIG. 9A, the order of peaks from left to right is:
Untreated, ImPACT, ImPACT plus OX40 antibody, and ComPACT. FIG. 9B
is a series of graphs plotting total numbers of MNC, CD4, CD8,
OT-I, and OT-II cells for mice either untreated or vaccinated with
ImPACT, ImPACT+OX40(ab), or ComPACT as in FIG. 9A, except in
peripheral lymph nodes. For each data set in FIG. 9B, the order of
peaks from left to right is: Untreated, ImPACT, ImPACT plus OX40
antibody, and ComPACT. FIG. 9C is a series of graphs plotting mRNA
expression for T cell activation genes (ACTB, IL2, and Perforin 1
(PRF1)). qRT-PCR revealed antigen specific OT-1 (CD8) activation in
mice treated with ComPACT, compared to non-specific activation of
both endogenous and antigen specific OT-1 CD8 cells in mice treated
with OX40(ab). Cells obtained from peritoneal washes in FIG. 8A
above were sorted into populations of OT-1.sup.+ and OT-1.sup.- CD8
cells. Total RNA was harvested, reverse transcribed, and analyzed
by qPCR. ACTB levels were consistent between cell populations and
treatments, serving as a control. IL2 levels were significantly
elevated in OT-1.sup.+ cells of mice treated with ImPACT,
ImPACT+OX40(ab), and ComPACT, indicating significant T cell
activation with all vaccines/combinations. Consistent with FIG. 8C,
levels of PRF1 were elevated non-specifically in both OT-1.sup.-
and OT-1.sup.+ CD8 fractions of mice treated with OX40(ab), while
only increasing in OT-1.sup.+ cells of ComPACT treated mice.
Plotted values represent the mean from a minimum of 3 mice and
error is SEM. For each data set in FIG. 9C, the order of histograms
from left to right is: ImPACT, ImPACT plus OX40 antibody, and
ComPACT.
[0023] FIGS. 10A-10C show that in tumor bearing mice, ComPACT (in
this figure, Gp96-Ig/OX40L-Fc) treatment resulted in the maximum
number of tumor invading lymphocytes and tumor regression. FIG. 10A
is a schematic of the experimental setup. BALB/C mice were
inoculated with 2.times.10.sup.5 CT26 cells sub-dermally,
indicating day 0. On days 6 and 11, mice were either unvaccinated
or vaccinated with ImPACT, ImPACT+OX86(ab), ComPACT, or OX86(ab)
alone. Vaccine treatments consisted of 1.times.10.sup.6 cells and
100 .mu.g of antibody. FIG. 10B is a graph plotting tumor area on
the indicated days following tumor inoculation on day 0 and is
plotted as the mean from a minimum of 5 experimental mice per
sample group, with error as SEM. FIG. 10C is a graph plotting tumor
area on day 21 of the study. For each data set in FIG. 10C, the
order of peaks from left to right is: No vaccine, CT26 only
control, OX40 antibody only, ImPACT, ImPACT plus OX40 antibody, and
ComPACT.
[0024] FIGS. 11A-11E show that ComPACT (in this figure,
Gp96-Ig/OX40L-Fc) treatment resulted in CD8+ specific tumor
infiltration, hindered tumor growth, increased overall survival and
significant tumor rejection in the CT26 colorectal carcinoma model.
In FIG. 11A, mice were inoculated on day 0 with 5.times.10.sup.5
CT26 tumor cells injected subcutaneously in the rear flank. Mice
were either untreated or vaccinated on days 4, 7 and 10 with CT26
parental cells, OX40(ab) alone, ImPACT alone, ImPACT+OX40(ab) or
ComPACT. A cohort of mice were sacrificed on day 12 for tumor
genetic analysis. Remaining mice were monitored for 30 days to
measure tumor area and overall survival. FIG. 11B depicts analysis
of day 12 tumor gene expression. Total RNA was isolated from
dissociated tumors, reverse transcribed and analyzed by qPCR.
Values were normalized to 18S mRNA and the first `Untreated` only
replicate was set at 1. For each data set in FIG. 11B, the order of
histograms from left to right is: Untreated, CT26 only control,
OX40 antibody only, ImPACT, ImPACT plus OX40 antibody, and ComPACT.
In FIG. 11C, AH1-tetramer/antigen specific CD8+ cells were analyzed
in treated mice. For each data set in FIG. 11C, the order of peaks
from left to right is: Untreated, CT26 only control, OX40 antibody
only, ImPACT, ImPACT plus OX40 antibody, and ComPACT. FIG. 11D
shows tumor area as measured daily for 21 days following initial
tumor inoculation. In FIG. 11E, overall survival was determined
over a 30 day time course. 80% of ComPACT treated mice survived
according to experimental criteria and 47% of mice (7 out of 15)
completely rejected established tumors. One OX40(ab) only treated
mouse rejected the tumor by day 24 and one ImPACT+OX40(ab) treated
mouse rejected by day 25.
[0025] FIGS. 12A-12D show that ComPACT (in this figure,
Gp96-Ig/OX40L-Fc) generates antigen-specific CD8+ expansion,
delayed tumor growth, increased overall survival and tumor
rejection in an aggressive B16.F10-ova melanoma model. In FIG. 12A,
mice were adoptively transferred with 5.times.10.sup.5 OT-I cells
on day -1, and then inoculated on day 0 with 5.times.10.sup.5
B16.F10-ova tumor cells injected subcutaneously in the rear flank.
Mice were either untreated or vaccinated on days 4, 7 and 10 with
B16.F10-ova parental cells, OX40(ab) alone, ImPACT alone,
ImPACT+OX40(ab) or ComPACT. FIG. 12B shows antigen-specific CD8+
(OT-I) expansion following treatment over a time-course of 25 days.
In FIG. 12C, tumor area was measured throughout a 25 day time
course following initial tumor inoculation. In FIG. 12D, overall
survival was determined over a 30 day time course. Approximately
78% of ComPACT treated mice survived and 11% of the ComPACT treated
mice completely rejected established tumors. Only the ComPACT
treated group had complete tumor rejecters: 1 out of 9 mice or
approximately 11%.
[0026] FIG. 13 is a graph plotting the OT-1 expansion time-course
(similar to FIGS. 5D and 6C), using FOXP3-RFP mice seeded with OT-1
(CD8) cells on day -1. OT-1/GFP cells were analyzed by flow
cytometry in mice treated with No Vaccine, Ova only control cells,
ComPACT (96-Ig/OX40L or gp96-Ig/TL1A) or ComPACT.sup.2
(96-Ig/OX40L+TL1A), over 46 days, with initial vaccination on day 0
and a boost on day 35. Plotted values represent the mean and error
is SEM. ComPACT.sup.2 (gp96-Ig/OX40L+TL1A) represents a combination
injection including ComPACT-OX40L and ComPACT-TL1A (i.e., two
different cell lines in the same syringe).
[0027] FIG. 14 is a graph showing the effects of ComPACT on the
proliferation and activation of ovalbumin specific CD8+ T cells
(OTI). C57BL/6 mice were immunized with ImPACT alone or ComPACT
(gp96-Ig/OX40L), ComPACT (96-Ig/4-ICOSL), or ComPACT
(gp96-Ig/4-1BBL) at day 0. The frequency of OT-I was monitored in
the peripheral blood on the indicated days.
[0028] FIG. 15 is a graph showing the effect of ComPACT on tumor
growth kinetics in the CT26 colorectal carcinoma model. Mice were
inoculated on day 0 with 5.times.10.sup.5 CT26 tumor cells injected
subcutaneously in the rear flank. Mice were either untreated or
vaccinated on days 4, 7 and 10 with CT26 parental cells, ImPACT
alone, ImPACT+the TNFRSF25 agonist (4C12 ab), 4C12 (ab) alone, PD-1
(ab) alone, 4C12 (ab) and PD-1 (ab), ComPACT (gp96-Ig/OX40L or
gp96-Ig/TL1A), ComPACT (gp96-Ig/OX40L)+PD-1 (ab), or ComPACT.sup.2
(gp96-Ig/OX40L+TL1A). The mice were monitored for 30 days to
measure tumor area. ComPACT.sup.2 (gp96-Ig/OX40L+TL1A) represents a
combination injection including ComPACT-OX40L and ComPACT-TL1A
(i.e., two different cell lines in the same syringe).
[0029] FIG. 16 is a graph showing the effect of ComPACT on overall
mice survival in the CT26 colorectal carcinoma model. Mice were
treated with CT26 tumor cells and vaccinated as described in FIG.
15.
[0030] FIG. 17 is a graph showing the amount of human OX40L
produced by a human prostate specific vaccine (HS-1020, PC-3 cell
line).
[0031] FIG. 18 is a graph showing the amount of human OX40L
produced by a human lung specific vaccine (HS-120, AD100 cell
line)
DETAILED DESCRIPTION
[0032] Various secretable proteins, i.e. vaccine proteins as
described herein, can be used to stimulate an immune response in
vivo. For example, secretable heat-shock protein gp96-Ig based
allogeneic cellular vaccines can achieve high-frequency polyclonal
CD8+ T cell responses to femto-molar concentrations of tumor
antigens through antigen cross-priming in vivo (Oizumi et al., J
Immunol 2007, 179(4):2310-2317) Multiple immunosuppressive
mechanisms elaborated by established tumors can dampen the activity
of this vaccine approach, however. To evaluate the potential
utility of combination immunotherapy for patients with advanced
disease, a systematic comparison of PD-1, PD-L1, CTLA-4, and LAG-3
blocking antibodies in mouse models of long-established B16-F10
melanoma was carried out (see, the Examples herein), demonstrating
superior combination between gp96-Ig vaccination and PD-1 blockade
as compared to other checkpoints. Synergistic anti-tumor benefits
may result from triple combinations of gp96-Ig vaccination, PD-1
blockade, and T cell costimulation using one or of an agonist of
OX40 (e.g., an OX40 ligand-Ig (OX40L-Ig) fusion, or a fragment
thereof that binds OX40), an agonist of inducible T-cell
costimulator (ICOS) (e.g., an ICOS ligand-Ig (ICOSL-Ig) fusion, or
a fragment thereof that binds ICOS), an agonist of CD40 (e.g., a
CD40L-Ig fusion protein, or fragment thereof), an agonist of CD27
(e.g. a CD70-Ig fusion protein or fragment thereof), an agonist of
4-1BB (e.g., a 4-1BB ligand-Ig (4-1BBL-Ig) fusion, or a fragment
thereof that binds 4-1BB), an agonist of TNFRSF25 (e.g., a TL1A-Ig
fusion, or a fragment thereof that binds TNFRSF25), or an agonist
of glucocorticoid-induced tumor necrosis factor receptor (GITR)
(e.g., a GITR ligand-Ig (GITRL-Ig) fusion, or a fragment thereof
that binds GITF). The enthusiasm for development of such triple
combinations is tempered by the anticipated cost of such therapies,
however. To circumvent this issue, vaccine protein expressing
vectors (e.g., gp96-Ig expressing vectors) were re-engineered to
simultaneously express T cell costimulatory protein (e.g.,
ICOSL-Ig, 4-1BBL-Ig, or OX40L-Ig), to provide a costimulatory
benefit without the need for an additional antibody therapy. The
re-engineered vectors are provided herein, as are methods for their
use. When gp96-Ig and these costimulatory fusion proteins were
secreted by allogeneic cell lines, enhanced activation of
antigen-specific CD8+ T cells was observed (see, the Examples
herein). Thus, combination immunotherapy can be achieved by vector
re-engineering to obviate the need for completely separate
vaccine/antibody/fusion protein regimens.
Vaccine Proteins
[0033] Vaccine proteins can induce immune responses that find use
in the present invention. In various embodiments, the present
invention provides expression vectors comprising a first nucleotide
sequence that encode a secretable vaccine protein and a second
nucleotide sequence that encode a T cell costimulatory fusion
protein. Compositions comprising the expression vectors of the
present invention are also provided. In various embodiments, such
compositions are utilized in methods of treating subjects to
stimulate immune responses in the subject including enhancing the
activation of antigen-specific T cells in the subject. The present
compositions find use in the treatment of various diseases
including cancer.
[0034] The heat shock protein (hsp) gp96, localized in the
endoplasmic reticulum (ER), serves as a chaperone for peptides on
their way to MHC class I and II molecules. Gp96 obtained from tumor
cells and used as a vaccine can induce specific tumor immunity,
presumably through the transport of tumor-specific peptides to
antigen-presenting cells (APCs) (J Immunol 1999,
163(10):5178-5182). For example, gp96-associated peptides are
cross-presented to CD8 cells by dendritic cells (DCs).
[0035] A vaccination system was developed for antitumor therapy by
transfecting a gp96-Ig G1-Fc fusion protein into tumor cells,
resulting in secretion of gp96-Ig in complex with chaperoned tumor
peptides (see, J Immunother 2008, 31(4):394-401, and references
cited therein). Parenteral administration of gp96-Ig secreting
tumor cells triggers robust, antigen-specific CD8 cytotoxic T
lymphocyte (CTL) expansion, combined with activation of the innate
immune system. Tumor-secreted gp96 causes the recruitment of DCs
and natural killer (NK) cells to the site of gp96 secretion, and
mediates DC activation. Further, the endocytic uptake of gp96 and
its chaperoned peptides triggers peptide cross presentation via
major MHC class I, as well as strong, cognate CD8 activation
independent of CD4 cells.
[0036] The vectors provided herein contain a first nucleotide
sequence that encodes a gp96-Ig fusion protein. The coding region
of human gp96 is 2,412 bases in length (SEQ ID NO:1), and encodes
an 803 amino acid protein (SEQ ID NO:2) that includes a 21 amino
acid signal peptide at the amino terminus, a potential
transmembrane region rich in hydrophobic residues, and an ER
retention peptide sequence at the carboxyl terminus (GENBANK.RTM.
Accession No. X15187; see, Maki et al., Proc Natl Acad Sci USA
1990, 87:5658-5562). The DNA and protein sequences of human gp96
follow:
TABLE-US-00001 (SEQ ID NO: 1)
atgagggccctgtgggtgctgggcctctgctgcgtcctgctgaccttcgg
gtcggtcagagctgacgatgaagttgatgtggatggtacagtagaagagg
atctgggtaaaagtagagaaggatcaaggacggatgatgaagtagtacag
agagaggaagaagctattcagttggatggattaaatgcatcacaaataag
agaacttagagagaagtcggaaaagtttgccttccaagccgaagttaaca
gaatgatgaaacttatcatcaattcattgtataaaaataaagagattacc
tgagagaactgatttcaaatgcttctgatgctttagataagataaggcta
atatcactgactgatgaaaatgctctactggaaatgaggaactaacagtc
aaaattaagtgtgataaggagaagaacctgctgcatgtcacagacaccgg
tgtaggaatgaccagagaagagttggttaaaaaccttggtaccatagcca
aatctgggacaagcgagtttttaaacaaaatgactgaagcacaggaagat
ggccagtcaacttctgaattgattggccagtaggtgtcggtactattccg
ccttccttgtagcagataaggttattgtcacttcaaaacacaacaacgat
acccagcacatctgggagtctgactccaatgaattttctgtaattgctga
cccaagaggaaacactctaggacggggaacgacaattacccttgtcttaa
aagaagaagcatctgattaccttgaattggatacaattaaaaatctcgtc
aaaaaatattcacagttcataaactacctatttatgtatggagcagcaag
actgaaactgagaggagcccatggaggaagaagaagcagccaaagaagag
aaagaagaatctgatgatgaagctgcagtagaggaagaagaagaagaaaa
gaaaccaaagactaaaaaagttgaaaaaactgtctgggactgggaactta
tgaatgatatcaaaccaatatggcagagaccatcaaaagaagtagaagaa
gatgaatacaaagctactacaaatcattttcaaaggaaagtgatgacccc
atggcttatattcactttactgctgaaggggaagttaccttcaaatcaat
tttatttgtacccacatctgctccacgtggtctgtagacgaatatggatc
taaaaagagcgattacattaagctctatgtgcgccgtgtattcatcacag
acgacttccatgatatgatgcctaaatacctcaattagtcaagggtgtgg
tggactcagatgatctccccttgaatgtttcccgcgagactcttcagcaa
cataaactgcttaaggtgattaggaagaagcttgacgtaaaacgctggac
atgatcaagaagattgctgatgataaatacaatgatactttttggaaaga
ataggtaccaacatcaagcttggtgtgattgaagaccactcgaatcgaac
acgtcttgctaaacttcttaggttccagtcttctcatcatccaactgaca
ttactagcctagaccagtatgtggaaagaatgaaggaaaaacaagacaaa
atctacttcatggctgggtccagcagaaaagaggctgaatcttctccatt
tgttgagcgacttctgaaaaagggctatgaagttatttacctcacagaac
ctgtggatgaatactgtattcaggcccttcccgaatttgatgggaagagg
accagaatgttgccaaggaaggagtgaagttcgatgaaagtgagaaaact
aaggagagtcgtgaagcagttgagaaagaatttgagcctctgctgaattg
gatgaaagataaagcccttaaggacaagattgaaaaggctgtggtgtctc
agcgcctgacagaatctccgtgtgctttggtggccagccagtacggatgg
tctggcaacatggagagaatcatgaaagcacaagcgtaccaaacgggcaa
ggacatctctacaaattactatgcgagtcagaagaaaacatttgaaatta
atcccagacacccgctgatcagagacatgcttcgacgaattaaggaagat
gaagatgataaaacagtatggatcttgctgtggattgatgaaacagcaac
gcttcggtcagggtatcttttaccagacactaaagcatatggagatagaa
tagaaagaatgcttcgcctcagtttgaacattgaccctgatgcaaaggtg
gaagaagagcccgaagaagaacctgaagagacagcagaagacacaacaga
agacacagagcaagacgaagatgaagaaatggatgtgggaacagatgaag
aagaagaaacagcaaaggaatctacagctgaaaaagatgaattgtaa (SEQ ID NO: 2)
MRALWVLGLCCVLLTFGSVRADDEVDVDGTVEEDLGKSREG
SRTDDEVVQREEEAIQLDGLNASQIRELREKSEKFAFQAEVNR
MMKLIINSLYKNKEIFLRELISNASDALDKIRLISLTDENALSG
NEELTVKIKCDKEKNLLHVTDTGVGMTREELVKNLGTIAKSG
TSEFLNKMTEAQEDGQSTSELIGQFGVGFYSAFLVADKVIVTS
KHNNDTQHIWESDSNEFSVIADPRGNTLGRGTTITLVLKEEAS
DYLELDTIKNLVKKYSQFINFPIYVWSSKTETVEEPMEEEEAA
KEEKEESDDEAAVEEEEEEKKPKTKKVEKTVWDWELMNDIK
PIWQRPSKEVEEDEYKAFYKSFSKESDDPMAYIHFTAEGEVTF
KSILFVPTSAPRGLFDEYGSKKSDYIKLYVRRVFITDDFHDMM
PKYLNFVKGVVDSDDLPLNVSRETLQQHKLLKVIRKKLVRKT
LDMIKKIADDKYNDTFWKEFGTNIKLGVIEDHSNRTRLAKLL
RFQSSHHPTDITSLDQYVERMKEKQDKIYFMAGSSRKEAESSP
FVERLLKKGYEVIYLTEPVDEYCIQALPEFDGKRFQNVAKEG
VKFDESEKTKESREAVEKEFEPLLNWMKDKALKDKIEKAVV
SQRLTESPCALVASQYGWSGNMERIMKAQAYQTGKDISTNY
YASQKKTFEINPRHPLIRDMLRRIKEDEDDKTVLDLAVVLFET
ATLRSGYLLPDTKAYGDRIERMLRLSLNIDPDAKVEEEPEEEP
EETAEDTTEDTEQDEDEEMDVGTDEEEETAKESTAEKDEL.
[0037] A nucleic acid encoding a gp96-Ig fusion sequence can be
produced using the methods described in U.S. Pat. No. 8,685,384,
which is incorporated herein by reference in its entirety. In some
embodiments, the gp96 portion of a gp96-Ig fusion protein can
contain all or a portion of a wild type gp96 sequence (e.g., the
human sequence set forth in SEQ ID NO:2). For example, a secretable
gp96-Ig fusion protein can include the first 799 amino acids of SEQ
ID NO:2, such that it lacks the C-terminal KDEL (SEQ ID NO:3)
sequence. Alternatively, the gp96 portion of the fusion protein can
have an amino acid sequence that contains one or more
substitutions, deletions, or additions as compared to the first 799
amino acids of the wild type gp96 sequence, such that it has at
least 90% (e.g., at least 90%, at least 91%, at least 92%, at least
93%, at least 94%, at least 95%, at least 96%, at least 97%, at
least 98%, or at least 99%) sequence identity to the wild type
polypeptide.
[0038] As used throughout this disclosure, the percent sequence
identity between a particular nucleic acid or amino acid sequence
and a sequence referenced by a particular sequence identification
number is determined as follows. First, a nucleic acid or amino
acid sequence is compared to the sequence set forth in a particular
sequence identification number using the BLAST 2 Sequences (B12seq)
program from the stand-alone version of BLASTZ containing BLASTN
version 2.0.14 and BLASTP version 2.0.14. This stand-alone version
of BLASTZ can be obtained online at fr.com/blast or at
ncbi.nlm.nih.gov. Instructions explaining how to use the Bl2seq
program can be found in the readme file accompanying BLASTZ. Bl2seq
performs a comparison between two sequences using either the BLASTN
or BLASTP algorithm. BLASTN is used to compare nucleic acid
sequences, while BLASTP is used to compare amino acid sequences. To
compare two nucleic acid sequences, the options are set as follows:
-i is set to a file containing the first nucleic acid sequence to
be compared (e.g., C:\seq1.txt); -j is set to a file containing the
second nucleic acid sequence to be compared (e.g., C:\seq2.txt); -p
is set to blastn; -o is set to any desired file name (e.g.,
C:\output.txt); -q is set to -1; -r is set to 2; and all other
options are left at their default setting. For example, the
following command can be used to generate an output file containing
a comparison between two sequences: C:\Bl2seq c:\seq1.txt -j
c:\seq2.txt -p blastn -o c:\output.txt -q -1 -r 2. To compare two
amino acid sequences, the options of Bl2seq are set as follows: -i
is set to a file containing the first amino acid sequence to be
compared (e.g., C:\seq1.txt); -j is set to a file containing the
second amino acid sequence to be compared (e.g., C:\seq2.txt); -p
is set to blastp; -o is set to any desired file name (e.g.,
C:\output.txt); and all other options are left at their default
setting. For example, the following command can be used to generate
an output file containing a comparison between two amino acid
sequences: C:\Bl2seq c: \seq1.txt -j c:\seg2.txt -p blastp -o
c:\output.txt. If the two compared sequences share homology, then
the designated output file will present those regions of homology
as aligned sequences. If the two compared sequences do not share
homology, then the designated output file will not present aligned
sequences.
[0039] Once aligned, the number of matches is determined by
counting the number of positions where an identical nucleotide or
amino acid residue is presented in both sequences. The percent
sequence identity is determined by dividing the number of matches
either by the length of the sequence set forth in the identified
sequence (e.g., SEQ ID NO:1), or by an articulated length (e.g.,
100 consecutive nucleotides or amino acid residues from a sequence
set forth in an identified sequence), followed by multiplying the
resulting value by 100. For example, a nucleic acid sequence that
has 2,200 matches when aligned with the sequence set forth in SEQ
ID NO:1 is 91.2 percent identical to the sequence set forth in SEQ
ID NO:1 (i.e., 2,000/2,412.times.100=91.2). It is noted that the
percent sequence identity value is rounded to the nearest tenth.
For example, 75.11, 75.12, 75.13, and 75.14 is rounded down to
75.1, while 75.15, 75.16, 75.17, 75.18, and 75.19 is rounded up to
75.2. It also is noted that the length value will always be an
integer.
[0040] Thus, in some embodiments, the gp96 portion of nucleic acid
encoding a gp96-Ig fusion polypeptide can encode an amino acid
sequence that differs from the wild type gp96 polypeptide at one or
more amino acid positions, such that it contains one or more
conservative substitutions, non-conservative substitutions, splice
variants, isoforms, homologues from other species, and
polymorphisms.
[0041] As defined herein, a "conservative substitution" denotes the
replacement of an amino acid residue by another, biologically
similar, residue. Typically, biological similarity, as referred to
above, reflects substitutions on the wild type sequence with
conserved amino acids. For example, conservative amino acid
substitutions would be expected to have little or no effect on
biological activity, particularly if they represent less than 10%
of the total number of residues in the polypeptide or protein.
Conservative substitutions may be made, for instance, on the basis
of similarity in polarity, charge, size, solubility,
hydrophobicity, hydrophilicity, and/or the amphipathic nature of
the amino acid residues involved. The 20 naturally occurring amino
acids can be grouped into the following six standard amino acid
groups: (1) hydrophobic: Met, Ala, Val, Leu, Ile; (2) neutral
hydrophilic: Cys, Ser, Thr; Asn, Gln; (3) acidic: Asp, Glu; (4)
basic: His, Lys, Arg; (5) residues that influence chain
orientation: Gly, Pro; and (6) aromatic: Trp, Tyr, Phe.
Accordingly, conservative substitutions may be effected by
exchanging an amino acid by another amino acid listed within the
same group of the six standard amino acid groups shown above. For
example, the exchange of Asp by Glu retains one negative charge in
the so modified polypeptide. In addition, glycine and proline may
be substituted for one another based on their ability to disrupt
.alpha.-helices. Additional examples of conserved amino acid
substitutions, include, without limitation, the substitution of one
hydrophobic residue for another, such as isoleucine, valine,
leucine, or methionine, or the substitution of one polar residue
for another, such as the substitution of arginine for lysine,
glutamic for aspartic acid, or glutamine for asparagine, and the
like. The term "conservative substitution" also includes the use of
a substituted amino acid residue in place of an un-substituted
parent amino acid residue, provided that antibodies raised to the
substituted polypeptide also immunoreact with the un-substituted
polypeptide.
[0042] As used herein, "non-conservative substitutions" are defined
as exchanges of an amino acid by another amino acid listed in a
different group of the six standard amino acid groups (1) to (6)
shown above.
[0043] In various embodiments, the substitutions may also include
non-classical amino acids (e.g. selenocysteine, pyrrolysine,
N-formylmethionine (.beta.-alanine, GABA and .delta.-Aminolevulinic
acid, 4-aminobenzoic acid (PABA), D-isomers of the common amino
acids, 2,4-diaminobutyric acid, .alpha.-amino isobutyric acid,
4-aminobutyric acid, Abu, 2-amino butyric acid, .gamma.-Abu,
.epsilon.-Ahx, 6-amino hexanoic acid, Aib, 2-amino isobutyric acid,
3-amino propionic acid, ornithine, norleucine, norvaline,
hydroxyproline, sarcosme, citrulline, homocitrulline, cysteic acid,
t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine,
.beta.-alanine, fluoro-amino acids, designer amino acids such as
.beta. methyl amino acids, C .alpha.-methyl amino acids, N
.alpha.-methyl amino acids, and amino acid analogs in general).
[0044] Mutations may also be made to the nucleotide sequences of
the present fusion proteins by reference to the genetic code,
including taking into account codon degeneracy.
[0045] The Ig portion ("tag") of a gp96-Ig fusion protein can
contain, for example, a non-variable portion of an immunoglobulin
molecule (e.g., an IgG1, IgG2, IgG3, IgG4, IgM, IgA, or IgE
molecule). Typically, such portions contain at least functional CH2
and CH3 domains of the constant region of an immunoglobulin heavy
chain Fusions also can be made using the carboxyl terminus of the
Fc portion of a constant domain, or a region immediately
amino-terminal to the CH1 of the heavy or light chain. The Ig tag
can be from a mammalian (e.g., human, mouse, monkey, or rat)
immunoglobulin, but human immunoglobulin can be particularly useful
when the 96-Ig fusion is intended for in vivo use for humans.
[0046] DNAs encoding immunoglobulin light or heavy chain constant
regions are known or readily available from cDNA libraries. See,
for example, Adams et al., Biochemistry 1980, 19:2711-2719; Gough
et al., Biochemistry 1980 19:2702-2710; Dolby et al., Proc Natl
Acad Sci USA 1980, 77:6027-6031; Rice et al., Proc Natl Acad Sci
USA 1982, 79:7862-7865; Falkner et al., Nature 1982, 298:286-288;
and Morrison et al., Ann Rev Immunol 1984, 2:239-256. Since many
immunological reagents and labeling systems are available for the
detection of immunoglobulins, gp96-Ig fusion proteins can readily
be detected and quantified by a variety of immunological techniques
known in the art, such as enzyme-linked immunosorbent assay
(ELISA), immunoprecipitation, and fluorescence activated cell
sorting (FACS). Similarly, if the peptide tag is an epitope with
readily available antibodies, such reagents can be used with the
techniques mentioned above to detect, quantitate, and isolate
gp96-Ig fusions.
[0047] In various embodiments, the 96-Ig fusion protein and/or the
costimulatory molecule fusions, comprises a linker In various
embodiments, the linker may be derived from naturally-occurring
multi-domain proteins or are empirical linkers as described, for
example, in Chichili et al., (2013), Protein Sci. 22(2):153-167,
Chen et al., (2013), Adv Drug Deliv Rev. 65(10):1357-1369, the
entire contents of which are hereby incorporated by reference. In
some embodiments, the linker may be designed using linker designing
databases and computer programs such as those described in Chen et
al., (2013), Adv Drug Deliv Rev. 65(10):1357-1369 and Crasto et.
al., (2000), Protein Eng. 13(5):309-312, the entire contents of
which are hereby incorporated by reference.
[0048] In some embodiments, the linker is a synthetic linker such
as PEG.
[0049] In other embodiments, the linker is a polypeptide. In some
embodiments, the linker is less than about 100 amino acids long.
For example, the linker may be less than about 100, about 95, about
90, about 85, about 80, about 75, about 70, about 65, about 60,
about 55, about 50, about 45, about 40, about 35, about 30, about
25, about 20, about 19, about 18, about 17, about 16, about 15,
about 14, about 13, about 12, about 11, about 10, about 9, about 8,
about 7, about 6, about 5, about 4, about 3, or about 2 amino acids
long. In some embodiments, the linker is flexible. In another
embodiment, the linker is rigid. In various embodiments, the linker
is substantially comprised of glycine and serine residues (e.g.
about 30%, or about 40%, or about 50%, or about 60%, or about 70%,
or about 80%, or about 90%, or about 95%, or about 97% glycines and
serines).
[0050] In various embodiments, the linker is a hinge region of an
antibody (e.g., of IgG, IgA, IgD, and IgE, inclusive of subclasses
(e.g. IgG1, IgG2, IgG3, and IgG4, and IgA1 and IgA2)). The hinge
region, found in IgG, IgA, IgD, and IgE class antibodies, acts as a
flexible spacer, allowing the Fab portion to move freely in space.
In contrast to the constant regions, the hinge domains are
structurally diverse, varying in both sequence and length among
immunoglobulin classes and subclasses. For example, the length and
flexibility of the hinge region varies among the IgG subclasses.
The hinge region of IgG1 encompasses amino acids 216-231 and,
because it is freely flexible, the Fab fragments can rotate about
their axes of symmetry and move within a sphere centered at the
first of two inter-heavy chain disulfide bridges. IgG2 has a
shorter hinge than IgG1, with 12 amino acid residues and four
disulfide bridges. The hinge region of IgG2 lacks a glycine
residue, is relatively short, and contains a rigid poly-proline
double helix, stabilized by extra inter-heavy chain disulfide
bridges. These properties restrict the flexibility of the IgG2
molecule. IgG3 differs from the other subclasses by its unique
extended hinge region (about four times as long as the IgG1 hinge),
containing 62 amino acids (including 21 prolines and 11 cysteines),
forming an inflexible poly-proline double helix. In IgG3, the Fab
fragments are relatively far away from the Fc fragment, giving the
molecule a greater flexibility. The elongated hinge in IgG3 is also
responsible for its higher molecular weight compared to the other
subclasses. The hinge region of IgG4 is shorter than that of IgG1
and its flexibility is intermediate between that of IgG1 and IgG2.
The flexibility of the hinge regions reportedly decreases in the
order IgG3>IgG1>IgG4>IgG2.
[0051] Additional illustrative linkers include, but are not limited
to, linkers having the sequence LE, GGGGS (SEQ ID NO:26),
(GGGGS).sub.n (n=1-4) (SEQ ID NO: 27), (Gly).sub.8 (SEQ ID NO:28),
(Gly).sub.6 (SEQ ID NO:29), (EAAAK).sub.n (n=1-3) (SEQ ID NO: 30),
A(EAAAK).sub.nA (n=2-5) (SEQ ID NO: 31), AEAAAKEAAAKA (SEQ ID NO:
32), A(EAAAK).sub.4ALEA(EAAAK).sub.4A (SEQ ID NO: 33), PAPAP (SEQ
ID NO: 34), KESGSVSSEQLAQFRSLD (SEQ ID NO: 35), EGKSSGSGSESKST(SEQ
ID NO: 36), GSAGSAAGSGEF (SEQ ID NO: 37), and (XP).sub.n, with X
designating any amino acid, e.g., Ala, Lys, or Glu.
[0052] In various embodiments, the linker may be functional. For
example, without limitation, the linker may function to improve the
folding and/or stability, improve the expression, improve the
pharmacokinetics, and/or improve the bioactivity of the present
compositions. In another example, the linker may function to target
the compositions to a particular cell type or location.
[0053] In some embodiments, a gp96 peptide can be fused to the
hinge, CH2 and CH3 domains of murine IgG1 (Bowen et al., J Immunol
1996, 156:442-449). This region of the IgG1 molecule contains three
cysteine residues that normally are involved in disulfide bonding
with other cysteines in the Ig molecule. Since none of the
cysteines are required for the peptide to function as a tag, one or
more of these cysteine residues can be substituted by another amino
acid residue, such as, for example, serine.
[0054] Various leader sequences known in the art also can be used
for efficient secretion of gp96-Ig fusion proteins from bacterial
and mammalian cells (see, von Heijne, J Mol Biol 1985, 184:99-105).
Leader peptides can be selected based on the intended host cell,
and may include bacterial, yeast, viral, animal, and mammalian
sequences. For example, the herpes virus glycoprotein D leader
peptide is suitable for use in a variety of mammalian cells.
Another leader peptide for use in mammalian cells can be obtained
from the V-J2-C region of the mouse immunoglobulin kappa chain
(Bernard et al., Proc Natl Acad Sci USA 1981, 78:5812-5816). DNA
sequences encoding peptide tags or leader peptides are known or
readily available from libraries or commercial suppliers, and are
suitable in the fusion proteins described herein.
[0055] Furthermore, in various embodiments, one may substitute the
gp96 of the present disclosure with one or more vaccine proteins.
For instance, various heat shock proteins are among the vaccine
proteins. In various embodiments, the heat shock protein is one or
more of a small hsp, hsp40, hsp60, hsp70, hsp90, and hsp110 family
member, inclusive of fragments, variants, mutants, derivatives or
combinations thereof (Hickey, et al., 1989, Mol. Cell. Biol.
9:2615-2626; Jindal, 1989, Mol. Cell. Biol. 9:2279-2283).
T-Cell Co-Stimulation
[0056] In addition to a gp96-Ig fusion protein, the expression
vectors provided herein can encode one or more biological response
modifiers. In various embodiments, the present expression vectors
can encode one or more T cell costimultory molecules.
[0057] In various embodiments, the present expression vectors allow
for a robust, antigen-specific CD8 cytotoxic T lymphocyte (CTL)
expansion. In various embodiments, the present expression vectors
selectively enhance CD8 cytotoxic T lymphocyte (CTL) and do not
substantially enhance T cell types that can be pro-tumor, and which
include, but are not limited to, Tregs, CD4+ and/or CD8+ T cells
expressing one or more checkpoint inhibitory receptors, Th2 cells
and Th17 cells. Checkpoint inhibitory receptors refers to receptors
(e.g. CTLA-4, B7-H3, B7-H4, TIM-3) expressed on immune cells that
prevent or inhibit uncontrolled immune responses. For instance, the
present expression vectors do not substantially enhance FOXP3.sup.+
regulatory T cells. In some embodiments, this selective CD8 T cell
enhancement is in contrast to the non-specific T cell enhancement
observed with a combination therapy of a gp-96 fusion and an
antibody against a T cell costimultory molecule.
[0058] For example, a vector can encode an agonist of OX40 (e.g.,
an OX40 ligand-Ig (OX40L-Ig) fusion, or a fragment thereof that
binds OX40), an agonist of inducible T-cell costimulator (ICOS)
(e.g., an ICOS ligand-Ig (ICOSL-Ig) fusion, or a fragment thereof
that binds ICOS), an agonist of CD40 (e.g., a CD40L-Ig fusion
protein, or fragment thereof), an agonist of CD27 (e.g. a CD70-Ig
fusion protein or fragment thereof), or an agonist of 4-1BB (e.g.,
a 4-1BB ligand-Ig (4-1BBL-Ig) fusion, or a fragment thereof that
binds 4-1BB). In some embodiments, a vector can encode an agonist
of TNFRSF25 (e.g., a TL1A-Ig fusion, or a fragment thereof that
binds TNFRSF25), or an agonist of glucocorticoid-induced tumor
necrosis factor receptor (GITR) (e.g., a GITR ligand-Ig (GITRL-Ig)
fusion, or a fragment thereof that binds GITR), or an agonist of
CD40 (e.g., a CD40 ligand-Ig (CD40L-Ig) fusion, or a fragment
thereof that binds CD40); or an agonist of CD27 (e.g., a CD27
ligand-Ig (e.g. CD70L-Ig) fusion, or a fragment thereof that binds
CD40).
[0059] ICOS is an inducible T cell costimulatory receptor molecule
that displays some homology to CD28 and CTLA-4, and interacts with
B7-H2 expressed on the surface of antigen-presenting cells. ICOS
has been implicated in the regulation of cell-mediated and humoral
immune responses.
[0060] 4-1BB is a type 2 transmembrane glycoprotein belonging to
the TNF superfamily, and is expressed on activated T
Lymphocytes.
[0061] OX40 (also referred to as CD134 or TNFRSF4) is a T cell
costimulatory molecule that is engaged by OX40L, and frequently is
induced in antigen presenting cells and other cell types. OX40 is
known to enhance cytokine expression and survival of effector T
cells.
[0062] GITR (TNFRSF18) is a T cell costimulatory molecule that is
engaged by GITRL and is preferentially expressed in FoxP3+
regulatory T cells. GITR plays a significant role in the
maintenance and function of Treg within the tumor
microenvironment.
[0063] TNFRSF25 is a T cell costimulatory molecule that is
preferentially expressed in CD4+ and CD8+ T cells following antigen
stimulation. Signaling through TNFRSF25 is provided by TL1A, and
functions to enhance T cell sensitivity to IL-2 receptor mediated
proliferation in a cognate antigen dependent manner.
[0064] CD40 is a costimulatory protein found on various antigen
presenting cells which plays a role in their activation. The
binding of CD40L (CD154) on T.sub.H cells to CD40 activates antigen
presenting cells and induces a variety of downstream effects.
[0065] CD27 a T cell costimulatory molecule belonging to the TNF
superfamily which plays a role in the generation and long-term
maintenance of T cell immunity. It binds to a ligand CD70 in
various immunological processes.
[0066] Additional costimulatory molecules that may be utilized in
the present invention include, but are not limited to, HVEM, CD28,
CD30, CD30L, CD40, CD70, LIGHT (CD258), B7-1, and B7-2.
[0067] As for the gp96-Ig fusions, the Ig portion ("tag") of the T
cell costimulatory fusion protein can contain, a non-variable
portion of an immunoglobulin molecule (e.g., an IgG1, IgG2, IgG3,
IgG4, IgM, IgA, or IgE molecule). As described above, such portions
typically contain at least functional CH2 and CH3 domains of the
constant region of an immunoglobulin heavy chain. In some
embodiments, a T cell costimulatory peptide can be fused to the
hinge, CH2 and CH3 domains of murine IgG1 (Bowen et al., J Immunol
1996, 156:442-449). The Ig tag can be from a mammalian (e.g.,
human, mouse, monkey, or rat) immunoglobulin, but human
immunoglobulin can be particularly useful when the fusion protein
is intended for in vivo use for humans. Again, DNAs encoding
immunoglobulin light or heavy chain constant regions are known or
readily available from cDNA libraries. Various leader sequences as
described above also can be used for secretion of T cell
costimulatory fusion proteins from bacterial and mammalian
cells.
[0068] A representative nucleotide optimized sequence (SEQ ID NO:4)
encoding the extracellular domain of human ICOSL fused to Ig, and
the amino acid sequence of the encoded fusion (SEQ ID NO:5) are
provided:
TABLE-US-00002 (SEQ ID NO: 4)
ATGAGACTGGGAAGCCCTGGCCTGCTGTTTCTGCTGTTCAG
CAGCCTGAGAGCCGACACCCAGGAAAAAGAAGTGCGGGC
CATGGTGGGAAGCGACGTGGAACTGAGCTGCGCCTGTCCT
GAGGGCAGCAGATTCGACCTGAACGACGTGTACGTGTACT
GGCAGACCAGCGAGAGCAAGACCGTCGTGACCTACCACAT
CCCCCAGAACAGCTCCCTGGAAAACGTGGACAGCCGGTAC
AGAAACCGGGCCCTGATGTCTCCTGCCGGCATGCTGAGAG
GCGACTTCAGCCTGCGGCTGTTCAACGTGACCCCCCAGGA
CGAGCAGAAATTCCACTGCCTGGTGCTGAGCCAGAGCCTG
GGCTTCCAGGAAGTGCTGAGCGTGGAAGTGACCCTGCACG
TGGCCGCCAATTTCAGCGTGCCAGTGGTGTCTGCCCCCCAC
AGCCCTTCTCAGGATGAGCTGACCTTCACCTGTACCAGCAT
CAACGGCTACCCCAGACCCAATGTGTACTGGATCAACAAG
ACCGACAACAGCCTGCTGGACCAGGCCCTGCAGAACGATA
CCGTGTTCCTGAACATGCGGGGCCTGTACGACGTGGTGTCC
GTGCTGAGAATCGCCAGAACCCCCAGCGTGAACATCGGCT
GCTGCATCGAGAACGTGCTGCTGCAGCAGAACCTGACCGT
GGGCAGCCAGACCGGCAACGACATCGGCGAGAGAGACAA
GATCACCGAGAACCCCGTGTCCACCGGCGAGAAGAATGCC
GCCACCTCTAAGTACGGCCCTCCCTGCCCTTCTTGCCCAGC
CCCTGAATTTCTGGGCGGACCCTCCGTGTTTCTGTTCCCCC
CAAAGCCCAAGGACACCCTGATGATCAGCCGGACCCCCGA
AGTGACCTGCGTGGTGGTGGATGTGTCCCAGGAAGATCCC
GAGGTGCAGTTCAATTGGTACGTGGACGGGGTGGAAGTGC
ACAACGCCAAGACCAAGCCCAGAGAGGAACAGTTCAACA
GCACCTACCGGGTGGTGTCTGTGCTGACCGTGCTGCACCAG
GATTGGCTGAGCGGCAAAGAGTACAAGTGCAAGGTGTCCA
GCAAGGGCCTGCCCAGCAGCATCGAAAAGACCATCAGCAA
CGCCACCGGCCAGCCCAGGGAACCCCAGGTGTACACACTG
CCCCCTAGCCAGGAAGAGATGACCAAGAACCAGGTGTCCC
TGACCTGTCTCGTGAAGGGCTTCTACCCCTCCGATATCGCC
GTGGAATGGGAGAGCAACGGCCAGCCAGAGAACAACTAC
AAGACCACCCCCCCAGTGCTGGACAGCGACGGCTCATTCT
TCCTGTACTCCCGGCTGACAGTGGACAAGAGCAGCTGGCA
GGAAGGCAACGTGTTCAGCTGCAGCGTGATGCACGAAGCC
CTGCACAACCACTACACCCAGAAGTCCCTGTCTCTGTCCCT GGGCAAATGA (SEQ ID NO: 5)
MRLGSPGLLFLLFSSLRADTQEKEVRAMVGSDVELSCACPEG
SRFDLNDVYVYWQTSESKTVVTYHIPQNSSLENVDSRYRNRA
LMSPAGMLRGDFSLRLFNVTPQDEQKFHCLVLSQSLGFQEVL
SVEVTLHVAANFSVPVVSAPHSPSQDELTFTCTSINGYPRPNV
YWINKTDNSLLDQALQNDTVFLNMRGLYDVVSVLRIARTPS
VNIGCCIENVLLQQNLTVGSQTGNDIGERDKITENPVSTGEKN
AATSKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVT
CVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYR
VVSVLTVLHQDWLSGKEYKCKVSSKGLPSSIEKTISNATGQPR
EPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQ
PENNYKTTPPVLDSDGSFFLYSRLTVDKSSWQEGNVFSCSVM HEALHNHYTQKSLSLSLGK.
[0069] A representative nucleotide optimized sequence (SEQ ID NO:6)
encoding the extracellular domain of human 4-1BBL fused to Ig, and
the encoded amino acid sequence (SEQ ID NO:7) are provided:
TABLE-US-00003 (SEQ ID NO: 6)
ATGTCTAAGTACGGCCCTCCCTGCCCTAGCTGCCCTGCCCC
TGAATTTCTGGGCGGACCCAGCGTGTTCCTGTTCCCCCCAA
AGCCCAAGGACACCCTGATGATCAGCCGGACCCCCGAAGT
GACCTGCGTGGTGGTGGATGTGTCCCAGGAAGATCCCGAG
GTGCAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACA
ACGCCAAGACCAAGCCCAGAGAGGAACAGTTCAACAGCA
CCTACCGGGTGGTGTCCGTGCTGACCGTGCTGCACCAGGAT
TGGCTGAGCGGCAAAGAGTACAAGTGCAAGGTGTCCAGCA
AGGGCCTGCCCAGCAGCATCGAGAAAACCATCAGCAACGC
CACCGGCCAGCCCAGGGAACCCCAGGTGTACACACTGCCC
CCTAGCCAGGAAGAGATGACCAAGAACCAGGTGTCCCTGA
CCTGTCTCGTGAAGGGCTTCTACCCCTCCGATATCGCCGTG
GAATGGGAGAGCAACGGCCAGCCTGAGAACAACTACAAG
ACCACCCCCCCAGTGCTGGACAGCGACGGCTCATTCTTCCT
GTACAGCAGACTGACCGTGGACAAGAGCAGCTGGCAGGA
AGGCAACGTGTTCAGCTGCAGCGTGATGCACGAGGCCCTG
CACAACCACTACACCCAGAAGTCCCTGTCTCTGAGCCTGG
GCAAGGCCTGTCCATGGGCTGTGTCTGGCGCTAGAGCCTCT
CCTGGATCTGCCGCCAGCCCCAGACTGAGAGAGGGACCTG
AGCTGAGCCCCGATGATCCTGCCGGACTGCTGGATCTGAG
ACAGGGCATGTTCGCCCAGCTGGTGGCCCAGAACGTGCTG
CTGATCGATGGCCCCCTGAGCTGGTACAGCGATCCTGGACT
GGCTGGCGTGTCACTGACAGGCGGCCTGAGCTACAAAGAG
GACACCAAAGAACTGGTGGTGGCCAAGGCCGGCGTGTACT
ACGTGTTCTTTCAGCTGGAACTGCGGAGAGTGGTGGCCGG
CGAAGGATCCGGCTCTGTGTCTCTGGCTCTGCATCTGCAGC
CCCTGAGATCTGCTGCTGGCGCTGCTGCTCTGGCCCTGACA
GTGGACCTGCCTCCTGCCTCTAGCGAGGCCAGAAACAGCG
CATTCGGGTTTCAAGGCAGACTGCTGCACCTGTCTGCCGGC
CAGAGACTGGGAGTGCATCTGCACACAGAGGCCAGAGCCA
GGCACGCCTGGCAGCTGACTCAGGGCGCTACAGTGCTGGG
CCTGTTCAGAGTGACCCCCGAGATTCCAGCCGGCCTGCCTA GCCCCAGATCCGAATGA (SEQ ID
NO: 7) MSKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCV
VVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRV
VSVLTVLHQDWLSGKEYKCKVSSKGLPSSIEKTISNATGQPRE
PQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQP
ENNYKTTPPVLDSDGSFFLYSRLTVDKSSWQEGNVFSCSVMH
EALHNHYTQKSLSLSLGKACPWAVSGARASPGSAASPRLREG
PELSPDDPAGLLDLRQGMFAQLVAQNVLLIDGPLSWYSDPGL
AGVSLTGGLSYKEDTKELVVAKAGVYYVFFQLELRRVVAGE
GSGSVSLALHLQPLRSAAGAAALALTVDLPPASSEARNSAFG
FQGRLLHLSAGQRLGVHLHTEARARHAWQLTQGATVLGLFR VTPEIPAGLPSPRSE.
[0070] A representative nucleotide optimized sequence (SEQ ID NO:8)
encoding the extracellular domain of human TL1A fused to Ig, and
the encoded amino acid sequence (SEQ ID NO:9) are provided:
TABLE-US-00004 (SEQ ID NO: 8)
ATGTCTAAGTACGGCCCTCCCTGCCCTAGCTGCCCTGCCCC
TGAATTTCTGGGCGGACCCAGCGTGTTCCTGTTCCCCCCAA
AGCCCAAGGACACCCTGATGATCAGCCGGACCCCCGAAGT
GACCTGCGTGGTGGTGGATGTGTCCCAGGAAGATCCCGAG
GTGCAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACA
ACGCCAAGACCAAGCCCAGAGAGGAACAGTTCAACAGCA
CCTACCGGGTGGTGTCCGTGCTGACCGTGCTGCACCAGGAT
TGGCTGAGCGGCAAAGAGTACAAGTGCAAGGTGTCCAGCA
AGGGCCTGCCCAGCAGCATCGAGAAAACCATCAGCAACGC
CACCGGCCAGCCCAGGGAACCCCAGGTGTACACACTGCCC
CCTAGCCAGGAAGAGATGACCAAGAACCAGGTGTCCCTGA
CCTGTCTCGTGAAGGGCTTCTACCCCTCCGATATCGCCGTG
GAATGGGAGAGCAACGGCCAGCCTGAGAACAACTACAAG
ACCACCCCCCCAGTGCTGGACAGCGACGGCTCATTCTTCCT
GTACAGCAGACTGACCGTGGACAAGAGCAGCTGGCAGGA
AGGCAACGTGTTCAGCTGCAGCGTGATGCACGAGGCCCTG
CACAACCACTACACCCAGAAGTCCCTGTCTCTGAGCCTGG
GCAAGATCGAGGGCCGGATGGATAGAGCCCAGGGCGAAG
CCTGCGTGCAGTTCCAGGCTCTGAAGGGCCAGGAATTCGC
CCCCAGCCACCAGCAGGTGTACGCCCCTCTGAGAGCCGAC
GGCGATAAGCCTAGAGCCCACCTGACAGTCGTGCGGCAGA
CCCCTACCCAGCACTTCAAGAATCAGTTCCCCGCCCTGCAC
TGGGAGCACGAACTGGGCCTGGCCTTCACCAAGAACAGAA
TGAACTACACCAACAAGTTTCTGCTGATCCCCGAGAGCGG
CGACTACTTCATCTACAGCCAAGTGACCTTCCGGGGCATGA
CCAGCGAGTGCAGCGAGATCAGACAGGCCGGCAGACCTAA
CAAGCCCGACAGCATCACCGTCGTGATCACCAAAGTGACC
GACAGCTACCCCGAGCCCACCCAGCTGCTGATGGGCACCA
AGAGCGTGTGCGAAGTGGGCAGCAACTGGTTCCAGCCCAT
CTACCTGGGCGCCATGTTTAGTCTGCAAGAGGGCGACAAG
CTGATGGTCAACGTGTCCGACATCAGCCTGGTGGATTACAC
CAAAGAGGACAAGACCTTCTTCGGCGCCTTTCTGCTCTGA (SEQ ID NO: 9)
MSKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCV
VVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRV
VSVLTVLHQDWLSGKEYKCKVSSKGLPSSIEKTISNATGQPRE
PQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQP
ENNYKTTPPVLDSDGSFFLYSRLTVDKSSWQEGNVFSCSVMH
EALHNHYTQKSLSLSLGKIEGRMDRAQGEACVQFQALKGQE
FAPSHQQVYAPLRADGDKPRAHLTVVRQTPTQHFKNQFPAL
HWEHELGLAFTKNRMNYTNKFLLIPESGDYFIYSQVTFRGMT
SECSEIRQAGRPNKPDSITVVITKVTDSYPEPTQLLMGTKSVCE
VGSNWFQPIYLGAMFSLQEGDKLMVNVSDISLVDYTKEDKTF FGAFLL.
[0071] A representative nucleotide optimized sequence (SEQ ID
NO:10) encoding human OX40L-Ig, and the encoded amino acid sequence
(SEQ ID NO:11) are provided:
TABLE-US-00005 (SEQ ID NO: 10)
ATGTCTAAGTACGGCCCTCCCTGCCCTAGCTGCCCTGCCCC
TGAATTTCTGGGCGGACCCAGCGTGTTCCTGTTCCCCCCAA
AGCCCAAGGACACCCTGATGATCAGCCGGACCCCCGAAGT
GACCTGCGTGGTGGTGGATGTGTCCCAGGAAGATCCCGAG
GTGCAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACA
ACGCCAAGACCAAGCCCAGAGAGGAACAGTTCAACAGCA
CCTACCGGGTGGTGTCCGTGCTGACCGTGCTGCACCAGGAT
TGGCTGAGCGGCAAAGAGTACAAGTGCAAGGTGTCCAGCA
AGGGCCTGCCCAGCAGCATCGAGAAAACCATCAGCAACGC
CACCGGCCAGCCCAGGGAACCCCAGGTGTACACACTGCCC
CCTAGCCAGGAAGAGATGACCAAGAACCAGGTGTCCCTGA
CCTGTCTCGTGAAGGGCTTCTACCCCTCCGATATCGCCGTG
GAATGGGAGAGCAACGGCCAGCCTGAGAACAACTACAAG
ACCACCCCCCCAGTGCTGGACAGCGACGGCTCATTCTTCCT
GTACAGCAGACTGACCGTGGACAAGAGCAGCTGGCAGGA
AGGCAACGTGTTCAGCTGCAGCGTGATGCACGAGGCCCTG
CACAACCACTACACCCAGAAGTCCCTGTCTCTGAGCCTGG
GCAAGATCGAGGGCCGGATGGATCAGGTGTCACACAGATA
CCCCCGGATCCAGAGCATCAAAGTGCAGTTTACCGAGTAC
AAGAAAGAGAAGGGCTTTATCCTGACCAGCCAGAAAGAG
GACGAGATCATGAAGGTGCAGAACAACAGCGTGATCATCA
ACTGCGACGGGTTCTACCTGATCAGCCTGAAGGGCTACTTC
AGTCAGGAAGTGAACATCAGCCTGCACTACCAGAAGGACG
AGGAACCCCTGTTCCAGCTGAAGAAAGTGCGGAGCGTGAA
CAGCCTGATGGTGGCCTCTCTGACCTACAAGGACAAGGTG
TACCTGAACGTGACCACCGACAACACCAGCCTGGACGACT
TCCACGTGAACGGCGGCGAGCTGATCCTGATTCACCAGAA CCCCGGCGAGTTCTGCGTGCTCTGA
(SEQ ID NO: 11) MSKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCV
VVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRV
VSVLTVLHQDWLSGKEYKCKVSSKGLPSSIEKTISNATGQPRE
PQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQP
ENNYKTTPPVLDSDGSFFLYSRLTVDKSSWQEGNVFSCSVMH
EALHNHYTQKSLSLSLGKIEGRMDQVSHRYPRIQSIKVQFTEY
KKEKGFILTSQKEDEIMKVQNNSVIINCDGFYLISLKGYFSQEV
NISLHYQKDEEPLFQLKKVRSVNSLMVASLTYKDKVYLNVTT
DNTSLDDFHVNGGELILIHQNPGEFCVL.
[0072] Representative nucleotide and amino acid sequences for human
TL1A are set forth in SEQ ID NO:12 and SEQ ID NO:13,
respectively:
TABLE-US-00006 (SEQ ID NO: 12)
TCCCAAGTAGCTGGGACTACAGGAGCCCACCACCACCCCC
GGCTAATTTTTTGTATTTTTAGTAGAGACGGGGTTTCACCG
TGTTAGCCAAGATGGTCTTGATCACCTGACCTCGTGATCCA
CCCGCCTTGGCCTCCCAAAGTGCTGGGATTACAGGCATGA
GCCACCGCGCCCGGCCTCCATTCAAGTCTTTATTGAATATC
TGCTATGTTCTACACACTGTTCTAGGTGCTGGGGATGCAAC
AGGGGACAAAATAGGCAAAATCCCTGTCCTTTTGGGGTTG
ACATTCTAGTGACTCTTCATGTAGTCTAGAAGAAGCTCAGT
GAATAGTGTCTGTGGTTGTTACCAGGGACACAATGACAGG
AACATTCTTGGGTAGAGTGAGAGGCCTGGGGAGGGAAGGG
TCTCTAGGATGGAGCAGATGCTGGGCAGTCTTAGGGAGCC
CCTCCTGGCATGCACCCCCTCATCCCTCAGGCCACCCCCGT
CCCTTGCAGGAGCACCCTGGGGAGCTGTCCAGAGCGCTGT
GCCGCTGTCTGTGGCTGGAGGCAGAGTAGGTGGTGTGCTG
GGAATGCGAGTGGGAGAACTGGGATGGACCGAGGGGAGG
CGGGTGAGGAGGGGGGCAACCACCCAACACCCACCAGCTG
CTTTCAGTGTTCTGGGTCCAGGTGCTCCTGGCTGGCCTTGT
GGTCCCCCTCCTGCTTGGGGCCACCCTGACCTACACATACC
GCCACTGCTGGCCTCACAAGCCCCTGGTTACTGCAGATGA
AGCTGGGATGGAGGCTCTGACCCCACCACCGGCCACCCAT
CTGTCACCCTTGGACAGCGCCCACACCCTTCTAGCACCTCC
TGACAGCAGTGAGAAGATCTGCACCGTCCAGTTGGTGGGT
AACAGCTGGACCCCTGGCTACCCCGAGACCCAGGAGGCGC
TCTGCCCGCAGGTGACATGGTCCTGGGACCAGTTGCCCAG
CAGAGCTCTTGGCCCCGCTGCTGCGCCCACACTCTCGCCAG
AGTCCCCAGCCGGCTCGCCAGCCATGATGCTGCAGCCGGG
CCCGCAGCTCTACGACGTGATGGACGCGGTCCCAGCGCGG
CGCTGGAAGGAGTTCGTGCGCACGCTGGGGCTGCGCGAGG
CAGAGATCGAAGCCGTGGAGGTGGAGATCGGCCGCTTCCG
AGACCAGCAGTACGAGATGCTCAAGCGCTGGCGCCAGCAG
CAGCCCGCGGGCCTCGGAGCCGTTTACGCGGCCCTGGAGC
GCATGGGGCTGGACGGCTGCGTGGAAGACTTGCGCAGCCG
CCTGCAGCGCGGCCCGTGACACGGCGCCCACTTGCCACCT
AGGCGCTCTGGTGGCCCTTGCAGAAGCCCTAAGTACGGTT
ACTTATGCGTGTAGACATTTTATGTCACTTATTAAGCCGCT
GGCACGGCCCTGCGTAGCAGCACCAGCCGGCCCCACCCCT
GCTCGCCCCTATCGCTCCAGCCAAGGCGAAGAAGCACGAA
CGAATGTCGAGAGGGGGTGAAGACATTTCTCAACTTCTCG
GCCGGAGTTTGGCTGAGATCGCGGTATTAAATCTGTGAAA GAAAACAAAACAAAACAA (SEQ ID
NO: 13) MEQRPRGCAAVAAALLLVLLGARAQGGTRSPRCDCAGDFHK
KIGLFCCRGCPAGHYLKAPCTEPCGNSTCLVCPQDTFLAWEN
HHNSECARCQACDEQASQVALENCSAVADTRCGCKPGWFVE
CQVSQCVSSSPFYCQPCLDCGALHRHTRLLCSRRDTDCGTCL
PGFYEHGDGCVSCPTPPPSLAGAPWGAVQSAVPLSVAGGRV
GVFWVQVLLAGLVVPLLLGATLTYTYRHCWPHKPLVTADEA
GMEALTPPPATHLSPLDSAHTLLAPPDSSEKICTVQLVGNSWT
PGYPETQEALCPQVTWSWDQLPSRALGPAAAPTLSPESPAGS
PAMMLQPGPQLYDVMDAVPARRWKEFVRTLGLREAEIEAVE
VEIGRFRDQQYEMLKRWRQQQPAGLGAVYAALERMGLDGC VEDLRSRLQRGP.
[0073] Representative nucleotide and amino acid sequences for human
HVEM are set forth in SEQ ID NO:38 (accession no. CR456909) and SEQ
ID NO:39, respectively (accession no. CR456909):
TABLE-US-00007 (SEQ ID NO: 38)
ATGGAGCCTCCTGGAGACTGGGGGCCTCCTCCCTGGAGAT
CCACCCCCAAAACCGACGTCTTGAGGCTGGTGCTGTATCTC
ACCTTCCTGGGAGCCCCCTGCTACGCCCCAGCTCTGCCGTC
CTGCAAGGAGGACGAGTACCCAGTGGGCTCCGAGTGCTGC
CCCAAGTGCAGTCCAGGTTATCGTGTGAAGGAGGCCTGCG
GGGAGCTGACGGGCACAGTGTGTGAACCCTGCCCTCCAGG
CACCTACATTGCCCACCTCAATGGCCTAAGCAAGTGTCTGC
AGTGCCAAATGTGTGACCCAGCCATGGGCCTGCGCGCGAG
CCGGAACTGCTCCAGGACAGAGAACGCCGTGTGTGGCTGC
AGCCCAGGCCACTTCTGCATCGTCCAGGACGGGGACCACT
GCGCCGCGTGCCGCGCTTACGCCACCTCCAGCCCGGGCCA
GAGGGTGCAGAAGGGAGGCACCGAGAGTCAGGACACCCT
GTGTCAGAACTGCCCCCCGGGGACCTTCTCTCCCAATGGGA
CCCTGGAGGAATGTCAGCACCAGACCAAGTGCAGCTGGCT
GGTGACGAAGGCCGGAGCTGGGACCAGCAGCTCCCACTGG
GTATGGTGGTTTCTCTCAGGGAGCCTCGTCATCGTCATTGT
TTGCTCCACAGTTGGCCTAATCATATGTGTGAAAAGAAGA
AAGCCAAGGGGTGATGTAGTCAAGGTGATCGTCTCCGTCC
AGCGGAAAAGACAGGAGGCAGAAGGTGAGGCCACAGTCA
TTGAGGCCCTGCAGGCCCCTCCGGACGTCACCACGGTGGC
CGTGGAGGAGACAATACCCTCATTCACGGGGAGGAGCCCA AACCATTAA (SEQ ID NO: 39)
MEPPGDWGPPPWRSTPKTDVLRLVLYLTFLGAPCYAPALPSC
KEDEYPVGSECCPKCSPGYRVKEACGELTGTVCEPCPPGTYIA
HLNGLSKCLQCQMCDPAMGLRASRNCSRTENAVCGCSPGHF
CIVQDGDHCAACRAYATSSPGQRVQKGGTESQDTLCQNCPP
GTFSPNGTLEECQHQTKCSWLVTKAGAGTSSSHWVWWFLSG
SLVIVIVCSTVGLIICVKRRKPRGDVVKVIVSVQRKRQEAEGE
ATVIEALQAPPDVTTVAVEETIPSFTGRSPNH.
[0074] Representative nucleotide and amino acid sequences for human
CD28 are set forth in SEQ ID NO:40 (accession no. NM_006139) and
SEQ ID NO:41, respectively:
TABLE-US-00008 (SEQ ID NO: 40)
TAAAGTCATCAAAACAACGTTATATCCTGTGTGAAATGCTG
CAGTCAGGATGCCTTGTGGTTTGAGTGCCTTGATCATGTGC
CCTAAGGGGATGGTGGCGGTGGTGGTGGCCGTGGATGACG
GAGACTCTCAGGCCTTGGCAGGTGCGTCTTTCAGTTCCCCT
CACACTTCGGGTTCCTCGGGGAGGAGGGGCTGGAACCCTA
GCCCATCGTCAGGACAAAGATGCTCAGGCTGCTCTTGGCTC
TCAACTTATTCCCTTCAATTCAAGTAACAGGAAACAAGATT
TTGGTGAAGCAGTCGCCCATGCTTGTAGCGTACGACAATG
CGGTCAACCTTAGCTGCAAGTATTCCTACAATCTCTTCTCA
AGGGAGTTCCGGGCATCCCTTCACAAAGGACTGGATAGTG
CTGTGGAAGTCTGTGTTGTATATGGGAATTACTCCCAGCAG
CTTCAGGTTTACTCAAAAACGGGGTTCAACTGTGATGGGA
AATTGGGCAATGAATCAGTGACATTCTACCTCCAGAATTTG
TATGTTAACCAAACAGATATTTACTTCTGCAAAATTGAAGT
TATGTATCCTCCTCCTTACCTAGACAATGAGAAGAGCAATG
GAACCATTATCCATGTGAAAGGGAAACACCTTTGTCCAAG
TCCCCTATTTCCCGGACCTTCTAAGCCCTTTTGGGTGCTGGT
GGTGGTTGGTGGAGTCCTGGCTTGCTATAGCTTGCTAGTAA
CAGTGGCCTTTATTATTTTCTGGGTGAGGAGTAAGAGGAGC
AGGCTCCTGCACAGTGACTACATGAACATGACTCCCCGCC
GCCCCGGGCCCACCCGCAAGCATTACCAGCCCTATGCCCC
ACCACGCGACTTCGCAGCCTATCGCTCCTGACACGGACGC
CTATCCAGAAGCCAGCCGGCTGGCAGCCCCCATCTGCTCA
ATATCACTGCTCTGGATAGGAAATGACCGCCATCTCCAGCC
GGCCACCTCAGGCCCCTGTTGGGCCACCAATGCCAATTTTT
CTCGAGTGACTAGACCAAATATCAAGATCATTTTGAGACTC
TGAAATGAAGTAAAAGAGATTTCCTGTGACAGGCCAAGTC
TTACAGTGCCATGGCCCACATTCCAACTTACCATGTACTTA
GTGACTTGACTGAGAAGTTAGGGTAGAAAACAAAAAGGG
AGTGGATTCTGGGAGCCTCTTCCCTTTCTCACTCACCTGCA
CATCTCAGTCAAGCAAAGTGTGGTATCCACAGACATTTTAG
TTGCAGAAGAAAGGCTAGGAAATCATTCCTTTTGGTTAAAT
GGGTGTTTAATCTTTTGGTTAGTGGGTTAAACGGGGTAAGT
TAGAGTAGGGGGAGGGATAGGAAGACATATTTAAAAACC
ATTAAAACACTGTCTCCCACTCATGAAATGAGCCACGTAGT
TCCTATTTAATGCTGTTTTCCTTTAGTTTAGAAATACATAGA
CATTGTCTTTTATGAATTCTGATCATATTTAGTCATTTTGAC
CAAATGAGGGATTTGGTCAAATGAGGGATTCCCTCAAAGC
AATATCAGGTAAACCAAGTTGCTTTCCTCACTCCCTGTCAT
GAGACTTCAGTGTTAATGTTCACAATATACTTTCGAAAGAA
TAAAATAGTTCTCCTACATGAAGAAAGAATATGTCAGGAA
ATAAGGTCACTTTATGTCAAAATTATTTGAGTACTATGGGA
CCTGGCGCAGTGGCTCATGCTTGTAATCCCAGCACTTTGGG
AGGCCGAGGTGGGCAGATCACTTGAGATCAGGACCAGCCT
GGTCAAGATGGTGAAACTCCGTCTGTACTAAAAATACAAA
ATTTAGCTTGGCCTGGTGGCAGGCACCTGTAATCCCAGCTG
CCCAAGAGGCTGAGGCATGAGAATCGCTTGAACCTGGCAG
GCGGAGGTTGCAGTGAGCCGAGATAGTGCCACAGCTCTCC
AGCCTGGGCGACAGAGTGAGACTCCATCTCAAACAACAAC
AACAACAACAACAACAACAACAAACCACAAAATTATTTGA
GTACTGTGAAGGATTATTTGTCTAACAGTTCATTCCAATCA
GACCAGGTAGGAGCTTTCCTGTTTCATATGTTTCAGGGTTG
CACAGTTGGTCTCTTTAATGTCGGTGTGGAGATCCAAAGTG
GGTTGTGGAAAGAGCGTCCATAGGAGAAGTGAGAATACTG
TGAAAAAGGGATGTTAGCATTCATTAGAGTATGAGGATGA
GTCCCAAGAAGGTTCTTTGGAAGGAGGACGAATAGAATGG
AGTAATGAAATTCTTGCCATGTGCTGAGGAGATAGCCAGC
ATTAGGTGACAATCTTCCAGAAGTGGTCAGGCAGAAGGTG
CCCTGGTGAGAGCTCCTTTACAGGGACTTTATGTGGTTTAG
GGCTCAGAGCTCCAAAACTCTGGGCTCAGCTGCTCCTGTAC
CTTGGAGGTCCATTCACATGGGAAAGTATTTTGGAATGTGT
CTTTTGAAGAGAGCATCAGAGTTCTTAAGGGACTGGGTAA
GGCCTGACCCTGAAATGACCATGGATATTTTTCTACCTACA
GTTTGAGTCAACTAGAATATGCCTGGGGACCTTGAAGAAT
GGCCCTTCAGTGGCCCTCACCATTTGTTCATGCTTCAGTTA
ATTCAGGTGTTGAAGGAGCTTAGGTTTTAGAGGCACGTAG
ACTTGGTTCAAGTCTCGTTAGTAGTTGAATAGCCTCAGGCA
AGTCACTGCCCACCTAAGATGATGGTTCTTCAACTATAAAA
TGGAGATAATGGTTACAAATGTCTCTTCCTATAGTATAATC
TCCATAAGGGCATGGCCCAAGTCTGTCTTTGACTCTGCCTA
TCCCTGACATTTAGTAGCATGCCCGACATACAATGTTAGCT
ATTGGTATTATTGCCATATAGATAAATTATGTATAAAAATT
AAACTGGGCAATAGCCTAAGAAGGGGGGAATATTGTAACA
CAAATTTAAACCCACTACGCAGGGATGAGGTGCTATAATA
TGAGGACCTTTTAACTTCCATCATTTTCCTGTTTCTTGAAAT
AGTTTATCTTGTAATGAAATATAAGGCACCTCCCACTTTTA
TGTATAGAAAGAGGTCTTTTAATTTTTTTTTAATGTGAGAA
GGAAGGGAGGAGTAGGAATCTTGAGATTCCAGATCGAAAA
TACTGTACTTTGGTTGATTTTTAAGTGGGCTTCCATTCCATG
GATTTAATCAGTCCCAAGAAGATCAAACTCAGCAGTACTT
GGGTGCTGAAGAACTGTTGGATTTACCCTGGCACGTGTGCC
ACTTGCCAGCTTCTTGGGCACACAGAGTTCTTCAATCCAAG
TTATCAGATTGTATTTGAAAATGACAGAGCTGGAGAGTTTT
TTGAAATGGCAGTGGCAAATAAATAAATACTTTTTTTTAAA
TGGAAAGACTTGATCTATGGTAATAAATGATTTTGTTTTCT
GACTGGAAAAATAGGCCTACTAAAGATGAATCACACTTGA
GATGTTTCTTACTCACTCTGCACAGAAACAAAGAAGAAAT
GTTATACAGGGAAGTCCGTTTTCACTATTAGTATGAACCAA
GAAATGGTTCAAAAACAGTGGTAGGAGCAATGCTTTCATA
GTTTCAGATATGGTAGTTATGAAGAAAACAATGTCATTTGC
TGCTATTATTGTAAGAGTCTTATAATTAATGGTACTCCTAT
AATTTTTGATTGTGAGCTCACCTATTTGGGTTAAGCATGCC
AATTTAAAGAGACCAAGTGTATGTACATTATGTTCTACATA
TTCAGTGATAAAATTACTAAACTACTATATGTCTGCTTTAA
ATTTGTACTTTAATATTGTCTTTTGGTATTAAGAAAGATAT
GCTTTCAGAATAGATATGCTTCGCTTTGGCAAGGAATTTGG
ATAGAACTTGCTATTTAAAAGAGGTGTGGGGTAAATCCTTG
TATAAATCTCCAGTTTAGCCTTTTTTGAAAAAGCTAGACTT
TCAAATACTAATTTCACTTCAAGCAGGGTACGTTTCTGGTT
TGTTTGCTTGACTTCAGTCACAATTTCTTATCAGACCAATG
GCTGACCTCTTTGAGATGTCAGGCTAGGCTTACCTATGTGT
TCTGTGTCATGTGAATGCTGAGAAGTTTGACAGAGATCCA
ACTTCAGCCTTGACCCCATCAGTCCCTCGGGTTAACTAACT
GAGCCACCGGTCCTCATGGCTATTTTAATGAGGGTATTGAT
GGTTAAATGCATGTCTGATCCCTTATCCCAGCCATTTGCAC
TGCCAGCTGGGAACTATACCAGACCTGGATACTGATCCCA
AAGTGTTAAATTCAACTACATGCTGGAGATTAGAGATGGT
GCCAATAAAGGACCCAGAACCAGGATCTTGATTGCTATAG
ACTTATTAATAATCCAGGTCAAAGAGAGTGACACACACTC
TCTCAAGACCTGGGGTGAGGGAGTCTGTGTTATCTGCAAG
GCCATTTGAGGCTCAGAAAGTCTCTCTTTCCTATAGATATA
TGCATACTTTCTGACATATAGGAATGTATCAGGAATACTCA
ACCATCACAGGCATGTTCCTACCTCAGGGCCTTTACATGTC
CTGTTTACTCTGTCTAGAATGTCCTTCTGTAGATGACCTGG
CTTGCCTCGTCACCCTTCAGGTCCTTGCTCAAGTGTCATCTT
CTCCCCTAGTTAAACTACCCCACACCCTGTCTGCTTTCCTTG
CTTATTTTTCTCCATAGCATTTTACCATCTCTTACATTAGAC
ATTTTTCTTATTTATTTGTAGTTTATAAGCTTCATGAGGCAA
GTAACTTTGCTTTGTTTCTTGCTGTATCTCCAGTGCCCAGAG
CAGTGCCTGGTATATAATAAATATTTATTGACTGAGTGAAA AAAAAAAAAAAAAA (SEQ ID
NO: 41) MLRLLLALNLFPSIQVTGNKILVKQSPMLVAYDNAVNLSCKY
SYNLFSREFRASLHKGLDSAVEVCVVYGNYSQQLQVYSKTGF
NCDGKLGNESVTFYLQNLYVNQTDIYFCKIEVMYPPPYLDNE
KSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLL
VTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAP PRDFAAYRS.
[0075] Representative nucleotide and amino acid sequences for human
CD30L are set forth in SEQ ID NO:42 (accession no. L09753) and SEQ
ID NO:43, respectively:
TABLE-US-00009 (SEQ ID NO: 42)
CCAAGTCACATGATTCAGGATTCAGGGGGAGAATCCTTCTT
GGAACAGAGATGGGCCCAGAACTGAATCAGATGAAGAGA
GATAAGGTGTGATGTGGGGAAGACTATATAAAGAATGGAC
CCAGGGCTGCAGCAAGCACTCAACGGAATGGCCCCTCCTG
GAGACACAGCCATGCATGTGCCGGCGGGCTCCGTGGCCAG
CCACCTGGGGACCACGAGCCGCAGCTATTTCTATTTGACCA
CAGCCACTCTGGCTCTGTGCCTTGTCTTCACGGTGGCCACT
ATTATGGTGTTGGTCGTTCAGAGGACGGACTCCATTCCCAA
CTCACCTGACAACGTCCCCCTCAAAGGAGGAAATTGCTCA
GAAGACCTCTTATGTATCCTGAAAAGAGCTCCATTCAAGA
AGTCATGGGCCTACCTCCAAGTGGCAAAGCATCTAAACAA
AACCAAGTTGTCTTGGAACAAAGATGGCATTCTCCATGGA
GTCAGATATCAGGATGGGAATCTGGTGATCCAATTCCCTG
GTTTGTACTTCATCATTTGCCAACTGCAGTTTCTTGTACAAT
GCCCAAATAATTCTGTCGATCTGAAGTTGGAGCTTCTCATC
AACAAGCATATCAAAAAACAGGCCCTGGTGACAGTGTGTG
AGTCTGGAATGCAAACGAAACACGTATACCAGAATCTCTC
TCAATTCTTGCTGGATTACCTGCAGGTCAACACCACCATAT
CAGTCAATGTGGATACATTCCAGTACATAGATACAAGCAC
CTTTCCTCTTGAGAATGTGTTGTCCATCTTCTTATACAGTAA
TTCAGACTGAACAGTTTCTCTTGGCCTTCAGGAAGAAAGCG
CCTCTCTACCATACAGTATTTCATCCCTCCAAACACTTGGG
CAAAAAGAAAACTTTAGACCAAGACAAACTACACAGGGTA
TTAAATAGTATACTTCTCCTTCTGTCTCTTGGAAAGATACA
GCTCCAGGGTTAAAAAGAGAGTTTTTAGTGAAGTATCTTTC
AGATAGCAGGCAGGGAAGCAATGTAGTGTGGTGGGCAGA
GCCCCACACAGAATCAGAAGGGATGAATGGATGTCCCAGC
CCAACCACTAATTCACTGTATGGTCTTGATCTATTTCTTCTG
TTTTGAGAGCCTCCAGTTAAAATGGGGCTTCAGTACCAGA
GCAGCTAGCAACTCTGCCCTAATGGGAAATGAAGGGGAGC
TGGGTGTGAGTGTTTACACTGTGCCCTTCACGGGATACTTC
TTTTATCTGCAGATGGCCTAATGCTTAGTTGTCCAAGTCGC
GATCAAGGACTCTCTCACACAGGAAACTTCCCTATACTGGC
AGATACACTTGTGACTGAACCATGCCCAGTTTATGCCTGTC
TGACTGTCACTCTGGCACTAGGAGGCTGATCTTGTACTCCA
TATGACCCCACCCCTAGGAACCCCCAGGGAAAACCAGGCT
CGGACAGCCCCCTGTTCCTGAGATGGAAAGCACAAATTTA
ATACACCACCACAATGGAAAACAAGTTCAAAGACTTTTAC
TTACAGATCCTGGACAGAAAGGGCATAATGAGTCTGAAGG
GCAGTCCTCCTTCTCCAGGTTACATGAGGCAGGAATAAGA
AGTCAGACAGAGACAGCAAGACAGTTAACAACGTAGGTA
AAGAAATAGGGTGTGGTCACTCTCAATTCACTGGCAAATG
CCTGAATGGTCTGTCTGAAGGAAGCAACAGAGAAGTGGGG
AATCCAGTCTGCTAGGCAGGAAAGATGCCTCTAAGTTCTTG
TCTCTGGCCAGAGGTGTGGTATAGAACCAGAAACCCATAT
CAAGGGTGACTAAGCCCGGCTTCCGGTATGAGAAATTAAA
CTTGTATACAAAATGGTTGCCAAGGCAACATAAAATTATA AGAATTC (SEQ ID NO: 43)
MDPGLQQALNGMAPPGDTAMHVPAGSVASHLGTTSRSYFYL
TTATLALCLVFTVATIMVLVVQRTDSIPNSPDNVPLKGGNCSE
DLLCILKRAPFKKSWAYLQVAKHLNKTKLSWNKDGILHGVR
YQDGNLVIQFPGLYFIICQLQFLVQCPNNSVDLKLELLINKHIK
KQALVTVCESGMQTKHVYQNLSQFLLDYLQVNTTISVNVDT
FQYIDTSTFPLENVLSIFLYSNSD.
[0076] Representative nucleotide and amino acid sequences for human
CD40 are set forth in SEQ ID NO:44 (accession no. NM_001250) and
SEQ ID NO:45, respectively:
TABLE-US-00010 (SEQ ID NO: 44)
TTTCCTGGGCGGGGCCAAGGCTGGGGCAGGGGAGTCAGCA
GAGGCCTCGCTCGGGCGCCCAGTGGTCCTGCCGCCTGGTCT
CACCTCGCTATGGTTCGTCTGCCTCTGCAGTGCGTCCTCTG
GGGCTGCTTGCTGACCGCTGTCCATCCAGAACCACCCACTG
CATGCAGAGAAAAACAGTACCTAATAAACAGTCAGTGCTG
TTCTTTGTGCCAGCCAGGACAGAAACTGGTGAGTGACTGC
ACAGAGTTCACTGAAACGGAATGCCTTCCTTGCGGTGAAA
GCGAATTCCTAGACACCTGGAACAGAGAGACACACTGCCA
CCAGCACAAATACTGCGACCCCAACCTAGGGCTTCGGGTC
CAGCAGAAGGGCACCTCAGAAACAGACACCATCTGCACCT
GTGAAGAAGGCTGGCACTGTACGAGTGAGGCCTGTGAGAG
CTGTGTCCTGCACCGCTCATGCTCGCCCGGCTTTGGGGTCA
AGCAGATTGCTACAGGGGTTTCTGATACCATCTGCGAGCCC
TGCCCAGTCGGCTTCTTCTCCAATGTGTCATCTGCTTTCGA
AAAATGTCACCCTTGGACAAGCTGTGAGACCAAAGACCTG
GTTGTGCAACAGGCAGGCACAAACAAGACTGATGTTGTCT
GTGGTCCCCAGGATCGGCTGAGAGCCCTGGTGGTGATCCC
CATCATCTTCGGGATCCTGTTTGCCATCCTCTTGGTGCTGGT
CTTTATCAAAAAGGTGGCCAAGAAGCCAACCAATAAGGCC
CCCCACCCCAAGCAGGAACCCCAGGAGATCAATTTTCCCG
ACGATCTTCCTGGCTCCAACACTGCTGCTCCAGTGCAGGAG
ACTTTACATGGATGCCAACCGGTCACCCAGGAGGATGGCA
AAGAGAGTCGCATCTCAGTGCAGGAGAGACAGTGAGGCTG
CACCCACCCAGGAGTGTGGCCACGTGGGCAAACAGGCAGT
TGGCCAGAGAGCCTGGTGCTGCTGCTGCTGTGGCGTGAGG
GTGAGGGGCTGGCACTGACTGGGCATAGCTCCCCGCTTCT
GCCTGCACCCCTGCAGTTTGAGACAGGAGACCTGGCACTG
GATGCAGAAACAGTTCACCTTGAAGAACCTCTCACTTCACC
CTGGAGCCCATCCAGTCTCCCAACTTGTATTAAAGACAGA
GGCAGAAGTTTGGTGGTGGTGGTGTTGGGGTATGGTTTAGT
AATATCCACCAGACCTTCCGATCCAGCAGTTTGGTGCCCAG
AGAGGCATCATGGTGGCTTCCCTGCGCCCAGGAAGCCATA
TACACAGATGCCCATTGCAGCATTGTTTGTGATAGTGAACA
ACTGGAAGCTGCTTAACTGTCCATCAGCAGGAGACTGGCT
AAATAAAATTAGAATATATTTATACAACAGAATCTCAAAA
ACACTGTTGAGTAAGGAAAAAAAGGCATGCTGCTGAATGA
TGGGTATGGAACTTTTTAAAAAAGTACATGCTTTTATGTAT
GTATATTGCCTATGGATATATGTATAAATACAATATGCATC
ATATATTGATATAACAAGGGTTCTGGAAGGGTACACAGAA
AACCCACAGCTCGAAGAGTGGTGACGTCTGGGGTGGGGAA GAAGGGTCTGGGGG (SEQ ID NO:
45) MVRLPLQCVLWGCLLTAVHPEPPTACREKQYLINSQCCSLCQ
PGQKLVSDCTEFTETECLPCGESEFLDTWNRETHCHQHKYCD
PNLGLRVQQKGTSETDTICTCEEGWHCTSEACESCVLHRSCSP
GFGVKQIATGVSDTICEPCPVGFFSNVSSAFEKCHPWTSCETK
DLVVQQAGTNKTDVVCGPQDRLRALVVIPIIFGILFAILLVLVF
IKKVAKKPTNKAPHPKQEPQEINFPDDLPGSNTAAPVQETLHG
CQPVTQEDGKESRISVQERQ.
[0077] Representative nucleotide and amino acid sequences for human
CD70 are set forth in SEQ ID NO:46 (accession no. NM_001252) and
SEQ ID NO:47, respectively:
TABLE-US-00011 (SEQ ID NO: 46)
CCAGAGAGGGGCAGGCTGGTCCCCTGACAGGTTGAAGCAA
GTAGACGCCCAGGAGCCCCGGGAGGGGGCTGCAGTTTCCT
TCCTTCCTTCTCGGCAGCGCTCCGCGCCCCCATCGCCCCTC
CTGCGCTAGCGGAGGTGATCGCCGCGGCGATGCCGGAGGA
GGGTTCGGGCTGCTCGGTGCGGCGCAGGCCCTATGGGTGC
GTCCTGCGGGCTGCTTTGGTCCCATTGGTCGCGGGCTTGGT
GATCTGCCTCGTGGTGTGCATCCAGCGCTTCGCACAGGCTC
AGCAGCAGCTGCCGCTCGAGTCACTTGGGTGGGACGTAGC
TGAGCTGCAGCTGAATCACACAGGACCTCAGCAGGACCCC
AGGCTATACTGGCAGGGGGGCCCAGCACTGGGCCGCTCCT
TCCTGCATGGACCAGAGCTGGACAAGGGGCAGCTACGTAT
CCATCGTGATGGCATCTACATGGTACACATCCAGGTGACG
CTGGCCATCTGCTCCTCCACGACGGCCTCCAGGCACCACCC
CACCACCCTGGCCGTGGGAATCTGCTCTCCCGCCTCCCGTA
GCATCAGCCTGCTGCGTCTCAGCTTCCACCAAGGTTGTACC
ATTGCCTCCCAGCGCCTGACGCCCCTGGCCCGAGGGGACA
CACTCTGCACCAACCTCACTGGGACACTTTTGCCTTCCCGA
AACACTGATGAGACCTTCTTTGGAGTGCAGTGGGTGCGCC
CCTGACCACTGCTGCTGATTAGGGTTTTTTAAATTTTATTTT
ATTTTATTTAAGTTCAAGAGAAAAAGTGTACACACAGGGG
CCACCCGGGGTTGGGGTGGGAGTGTGGTGGGGGGTAGTGG
TGGCAGGACAAGAGAAGGCATTGAGCTTTTTCTTTCATTTT CCTATTAAAAAATACAAAAATCA
(SEQ ID NO: 47) MPEEGSGCSVRRRPYGCVLRAALVPLVAGLVICLVVCIQRFA
QAQQQLPLESLGWDVAELQLNHTGPQQDPRLYWQGGPALG
RSFLHGPELDKGQLRIHRDGIYMVHIQVTLAICSSTTASRHHP
TTLAVGICSPASRSISLLRLSFHQGCTIASQRLTPLARGDTLCT
NLTGTLLPSRNTDETFFGVQWVRP.
[0078] Representative nucleotide and amino acid sequences for human
LIGHT are set forth in SEQ ID NO:48 (accession no. CR541854) and
SEQ ID NO:49, respectively:
TABLE-US-00012 (SEQ ID NO: 48)
ATGGAGGAGAGTGTCGTACGGCCCTCAGTGTTTGTGGTGG
ATGGACAGACCGACATCCCATTCACGAGGCTGGGACGAAG
CCACCGGAGACAGTCGTGCAGTGTGGCCCGGGTGGGTCTG
GGTCTCTTGCTGTTGCTGATGGGGGCCGGGCTGGCCGTCCA
AGGCTGGTTCCTCCTGCAGCTGCACTGGCGTCTAGGAGAG
ATGGTCACCCGCCTGCCTGACGGACCTGCAGGCTCCTGGG
AGCAGCTGATACAAGAGCGAAGGTCTCACGAGGTCAACCC
AGCAGCGCATCTCACAGGGGCCAACTCCAGCTTGACCGGC
AGCGGGGGGCCGCTGTTATGGGAGACTCAGCTGGGCCTGG
CCTTCCTGAGGGGCCTCAGCTACCACGATGGGGCCCTTGTG
GTCACCAAAGCTGGCTACTACTACATCTACTCCAAGGTGCA
GCTGGGCGGTGTGGGCTGCCCGCTGGGCCTGGCCAGCACC
ATCACCCACGGCCTCTACAAGCGCACACCCCGCTACCCCG
AGGAGCTGGAGCTGTTGGTCAGCCAGCAGTCACCCTGCGG
ACGGGCCACCAGCAGCTCCCGGGTCTGGTGGGACAGCAGC
TTCCTGGGTGGTGTGGTACACCTGGAGGCTGGGGAGGAGG
TGGTCGTCCGTGTGCTGGATGAACGCCTGGTTCGACTGCGT
GATGGTACCCGGTCTTACTTCGGGGCTTTCATGGTGTGA (SEQ ID NO: 49)
MEESVVRPSVFVVDGQTDIPFTRLGRSHRRQSCSVARVGLGL
LLLLMGAGLAVQGWFLLQLHWRLGEMVTRLPDGPAGSWEQ
LIQERRSHEVNPAAHLTGANSSLTGSGGPLLWETQLGLAFLR
GLSYHDGALVVTKAGYYYIYSKVQLGGVGCPLGLASTITHGL
YKRTPRYPEELELLVSQQSPCGRATSSSRVWWDSSFLGGVVH
LEAGEEVVVRVLDERLVRLRDGTRSYFGAFMV.
[0079] In various embodiments, the present invention provides for
variants comprising any of the sequences described herein, for
instance, a sequence having at least about 60%, or at least about
61%, or at least about 62%, or at least about 63%, or at least
about 64%, or at least about 65%, or at least about 66%, or at
least about 67%, or at least about 68%, or at least about 69%, or
at least about 70%, or at least about 71%, or at least about 72%,
or at least about 73%, or at least about 74%, or at least about
75%, or at least about 76%, or at least about 77%, or at least
about 78%, or at least about 79%, or at least about 80%, or at
least about 81%, or at least about 82%, or at least about 83%, or
at least about 84%, or at least about 85%, or at least about 86%,
or at least about 87%, or at least about 88%, or at least about
89%, or at least about 90%, or at least about 91%, or at least
about 92%, or at least about 93%, or at least about 94%, or at
least about 95%, or at least about 96%, or at least about 97%, or
at least about 98%, or at least about 99%) sequence identity with
any of the sequences disclosed herein (for example, SEQ ID NOS:
1-13 and 38-49).
[0080] In various embodiments, the present invention provides for
an amino acid sequence having one or more amino acid mutations
relative any of the protein sequences described herein. In some
embodiments, the one or more amino acid mutations may be
independently selected from conservative or non-conservative
substitutions, insertions, deletions, and truncations as described
herein.
Checkpoint Blockade/Blockage of Tumor Immunosuppression
[0081] Some human tumors can be eliminated by a patient's immune
system. For example, administration of a monoclonal antibody
targeted to an immune "checkpoint" molecule can lead to complete
response and tumor remission. A mode of action of such antibodies
is through inhibition of an immune regulatory molecule that the
tumors have co-opted as protection from an anti-tumor immune
response. By inhibiting these "checkpoint" molecules (e.g., with an
antagonistic antibody), a patient's CD8+ T cells may be allowed to
proliferate and destroy tumor cells.
[0082] For example, administration of a monoclonal antibody
targeted to by way of example, without limitation, CTLA-4 or PD-1
can lead to complete response and tumor remission. The mode of
action of such antibodies is through inhibition of CTLA-4 or PD-1
that the tumors have co-opted as protection from an anti-tumor
immune response. By inhibiting these "checkpoint" molecules (e.g.,
with an antagonistic antibody), a patient's CD8+ T cells may be
allowed to proliferate and destroy tumor cells.
[0083] Thus, the vectors provided herein can be used in combination
with one or more blocking antibodies targeted to an immune
"checkpoint" molecule. For instance, in some embodiments, the
vectors provided herein can be used in combination with one or more
blocking antibodies targeted to a molecule such as CTLA-4 or PD-1.
For example, the vectors provided herein may be used in combination
with an agent that blocks, reduces and/or inhibits PD-1 and PD-L1
or PD-L2 and/or the binding of PD-1 with PD-L1 or PD-L2 (by way of
non-limiting example, one or more of nivolumab
(ONO-4538/BMS-936558, MDX1106, OPDIVO, BRISTOL MYERS SQUIBB),
pembrolizumab (KEYTRUDA, Merck), pidilizumab (CT-011, CURE TECH),
MK-3475 (MERCK), BMS 936559 (BRISTOL MYERS SQUIBB), MPDL328OA
(ROCHE)). In an embodiment, the vectors provided herein may be used
in combination with an agent that blocks, reduces and/or inhibits
the activity of CTLA-4 and/or the binding of CTLA-4 with one or
more receptors (e.g. CD80, CD86, AP2M1, SHP-2, and PPP2R5A). For
instance, in some embodiments, the immune-modulating agent is an
antibody such as, by way of non-limitation, ipilimumab (MDX-010,
MDX-101, Yervoy, BMS) and/or tremelimumab (Pfizer). Blocking
antibodies against these molecules can be obtained from, for
example, Bristol Myers Squibb (New York, N.Y.), Merck (Kenilworth,
N.J.), Medlmmune (Gaithersburg, Md.), and Pfizer (New York,
N.Y.).
[0084] Further, the vectors provided herein can be used in
combination with one or more blocking antibodies targeted to an
immune "checkpoint" molecule such as for example, BTLA, HVEM, TIM3,
GALS, LAG3, VISTA, KIR, 2B4, CD160 (also referred to as BY55),
CGEN-15049, CHK 1 and CHK2 kinases, A2aR, CEACAM (e.g., CEACAM-1,
CEACAM-3 and/or CEACAM-5), GITR, GITRL, galectin-9, CD244, CD160,
TIGIT, SIRP.alpha., ICOS, CD172a, and TMIGD2 and various B-7 family
ligands (including, but are not limited to, B7-1, B7-2, B7-DC,
B7-H1, B7-H2, B7-H3, B7-H4, B7-H5, B7-H6 and B7-H7).
Vectors and Host Cells
[0085] This document provides nucleic acid constructs that encode a
vaccine protein fusion protein (e.g., a gp96-Ig fusion protein) and
a T cell costimulatory fusion protein that can be expressed in
prokaryotic and eukaryotic cells. For example, this document
provides expression vectors (e.g., DNA- or RNA-based vectors)
containing nucleotide sequences that encode a vaccine protein
fusion (e.g., a gp96-Ig fusion) and a T cell costimulatory fusion
protein (e.g., OX40L-Ig or a portion thereof that binds
specifically to OX40, ICOSL-Ig or a portion thereof that binds
specifically to ICOS, 4-1BBL-Ig, or a portion thereof that binds
specifically to 4-1BBR, CD40L-Ig, or a portion thereof that binds
specifically to CD40, CD70-Ig, or a portion thereof that binds
specifically to CD27, TL1A-Ig or a portion thereof that binds
specifically to TNFRSF25, or GITRL-Ig or a portion thereof that
binds specifically to GITR). In addition, this document provides
methods for making the vectors described herein, as well as methods
for introducing the vectors into appropriate host cells for
expression of the encoded polypeptides. In general, the methods
provided herein include constructing nucleic acid sequences
encoding a vaccine protein fusion protein (e.g., a gp96-Ig fusion
protein) and a T cell costimulatory fusion protein, cloning the
sequences encoding the fusion proteins into an expression vector.
The expression vector can be introduced into host cells or
incorporated into virus particles, either of which can be
administered to a subject to, for example, treat cancer or
infection. For example, gp96-Ig based vaccines can be generated to
stimulate antigen specific immune responses against individual
antigens expressed by simian immunodeficiency virus, human
immunodeficiency virus, hepatitis C virus and malaria Immune
responses to these vaccines may be enhanced through co-expression
of a T cell costimulatory fusion protein by the 96-Ig vector.
[0086] cDNA or DNA sequences encoding a vaccine protein fusion
(e.g., a gp96-Ig fusion) and a T cell costimulatory fusion protein
can be obtained (and, if desired, modified) using conventional DNA
cloning and mutagenesis methods, DNA amplification methods, and/or
synthetic methods. In general, a sequence encoding a vaccine
protein fusion protein (e.g., a gp96-Ig fusion protein) and/or a T
cell costimulatory fusion protein can be inserted into a cloning
vector for genetic modification and replication purposes prior to
expression. Each coding sequence can be operably linked to a
regulatory element, such as a promoter, for purposes of expressing
the encoded protein in suitable host cells in vitro and in
vivo.
[0087] Expression vectors can be introduced into host cells for
producing secreted vaccine proteins (e.g., gp96-Ig) and T cell
costimulatory fusion proteins. There are a variety of techniques
available for introducing nucleic acids into viable cells.
Techniques suitable for the transfer of nucleic acid into mammalian
cells in vitro include the use of liposomes, electroporation,
microinjection, cell fusion, polymer-based systems, DEAE-dextran,
viral transduction, the calcium phosphate precipitation method,
etc. For in vivo gene transfer, a number of techniques and reagents
may also be used, including liposomes; natural polymer-based
delivery vehicles, such as chitosan and gelatin; viral vectors are
also suitable for in vivo transduction. In some situations it is
desirable to provide a targeting agent, such as an antibody or
ligand specific for a cell surface membrane protein. Where
liposomes are employed, proteins which bind to a cell surface
membrane protein associated with endocytosis may be used for
targeting and/or to facilitate uptake, e.g., capsid proteins or
fragments thereof tropic for a particular cell type, antibodies for
proteins which undergo internalization in cycling, proteins that
target intracellular localization and enhance intracellular
half-life. The technique of receptor-mediated endocytosis is
described, for example, by Wu et al., J. Biol. Chem. 262, 4429-4432
(1987); and Wagner et al., Proc. Natl. Acad. Sci. USA 87, 3410-3414
(1990).
[0088] Where appropriate, gene delivery agents such as, e.g.,
integration sequences can also be employed. Numerous integration
sequences are known in the art (see, e.g., Nunes-Duby et al.,
Nucleic Acids Res. 26:391-406, 1998; Sadwoski, J. Bacteriol.,
165:341-357, 1986; Bestor, Cell, 122(3):322-325, 2005; Plasterk et
al., TIG 15:326-332, 1999; Kootstra et al., Ann. Rev. Pharm.
Toxicol., 43:413-439, 2003). These include recombinases and
transposases. Examples include Cre (Sternberg and Hamilton, J. Mol.
Biol., 150:467-486, 1981), lambda (Nash, Nature, 247, 543-545,
1974), FIp (Broach, et al., Cell, 29:227-234, 1982), R (Matsuzaki,
et al., J. Bacteriology, 172:610-618, 1990), cpC31 (see, e.g.,
Groth et al., J. Mol. Biol. 335:667-678, 2004), sleeping beauty,
transposases of the mariner family (Plasterk et al., supra), and
components for integrating viruses such as AAV, retroviruses, and
antiviruses having components that provide for virus integration
such as the LTR sequences of retroviruses or lentivirus and the ITR
sequences of AAV (Kootstra et al., Ann. Rev. Pharm. Toxicol.,
43:413-439, 2003).
[0089] Cells may be cultured in vitro or genetically engineered,
for example. Host cells can be obtained from normal or affected
subjects, including healthy humans, cancer patients, and patients
with an infectious disease, private laboratory deposits, public
culture collections such as the American Type Culture Collection,
or from commercial suppliers.
[0090] Cells that can be used for production and secretion of
gp96-Ig fusion proteins and T cell costimulatory fusion proteins in
vivo include, without limitation, epithelial cells, endothelial
cells, keratinocytes, fibroblasts, muscle cells, hepatocytes; blood
cells such as T lymphocytes, B lymphocytes, monocytes, macrophages,
neutrophils, eosinophils, megakaryocytes, or granulocytes, various
stem or progenitor cells, such as hematopoietic stem or progenitor
cells (e.g., as obtained from bone marrow), umbilical cord blood,
peripheral blood, fetal liver, etc., and tumor cells (e.g., human
tumor cells). The choice of cell type depends on the type of tumor
or infectious disease being treated or prevented, and can be
determined by one of skill in the art.
[0091] Different host cells have characteristic and specific
mechanisms for post-translational processing and modification of
proteins. A host cell may be chosen which modifies and processes
the expressed gene products in a specific fashion similar to the
way the recipient processes its heat shock proteins (hsps). For the
purpose of producing large amounts of gp96-Ig, it can be preferable
that the type of host cell has been used for expression of
heterologous genes, and is reasonably well characterized and
developed for large-scale production processes. In some
embodiments, the host cells are autologous to the patient to whom
the present fusion or recombinant cells secreting the present
fusion proteins are subsequently administered.
[0092] In some embodiments, an expression construct as provided
herein can be introduced into an antigenic cell. As used herein,
antigenic cells can include preneoplastic cells that are infected
with a cancer-causing infectious agent, such as a virus, but that
are not yet neoplastic, or antigenic cells that have been exposed
to a mutagen or cancer-causing agent, such as a DNA-damaging agent
or radiation, for example. Other cells that can be used are
preneoplastic cells that are in transition from a normal to a
neoplastic form as characterized by morphology or physiological or
biochemical function.
[0093] Typically, the cancer cells and preneoplastic cells used in
the methods provided herein are of mammalian origin Mammals
contemplated include humans, companion animals (e.g., dogs and
cats), livestock animals (e.g., sheep, cattle, goats, pigs and
horses), laboratory animals (e g., mice, rats and rabbits), and
captive or free wild animals.
[0094] In some embodiments, cancer cells (e.g., human tumor cells)
can be used in the methods described herein. The cancer cells
provide antigenic peptides that become associated non-covalently
with the expressed gp96-Ig fusion proteins. Cell lines derived from
a preneoplastic lesion, cancer tissue, or cancer cells also can be
used, provided that the cells of the cell line have at least one or
more antigenic determinant in common with the antigens on the
target cancer cells. Cancer tissues, cancer cells, cells infected
with a cancer-causing agent, other preneoplastic cells, and cell
lines of human origin can be used. Cancer cells excised from the
patient to whom ultimately the fusion proteins ultimately are to be
administered can be particularly useful, although allogeneic cells
also can be used. In some embodiments, a cancer cell can be from an
established tumor cell line such as, without limitation, an
established non-small cell lung carcinoma (NSCLC), bladder cancer,
melanoma, ovarian cancer, renal cell carcinoma, prostate carcinoma,
sarcoma, breast carcinoma, squamous cell carcinoma, head and neck
carcinoma, hepatocellular carcinoma, pancreatic carcinoma, or colon
carcinoma cell line.
[0095] In various embodiments, the present fusion proteins allow
for both the costimulation T cell and the presentation of various
tumor cell antigens. For instance, in some embodiments, the present
vaccine protein fusions (e.g., gp96 fusions) chaperone these
various tumor antigens. In various embodiments, the tumor cells
secrete a variety of antigens. Illustrative, but non-limiting,
antigens that can be secreted are: MART-1/Melan-A, gp100,
Dipeptidyl peptidase IV (DPPIV), adenosine deaminase-binding
protein (ADAbp), cyclophilin b, Colorectal associated antigen
(CRC)-0017-1A/GA733, Carcinoembryonic Antigen (CEA) and its
immunogenic epitopes CAP-1 and CAP-2, etv6, amll, Prostate Specific
Antigen (PSA) and its immunogenic epitopes PSA-1, PSA-2, and PSA-3,
prostate-specific membrane antigen (PSMA), T-cell receptor/CD3-zeta
chain, MAGE-family of tumor antigens (e.g., MAGE-A1, MAGE-A2,
MAGE-A3, MAGE-A4, MAGE-A5, MAGE-A6, MAGE-A7, MAGE-A8, MAGE-A9,
MAGE-A10, MAGE-A11, MAGE-A12, MAGE-Xp2 (MAGE-B2), MAGE-Xp3
(MAGE-B3), MAGE-Xp4 (MAGE-B4), MAGE-C1, MAGE-C2, MAGE-C3, MAGE-C4,
MAGE-C5), GAGE-family of tumor antigens (e.g., GAGE-1, GAGE-2,
GAGE-3, GAGE-4, GAGE-5, GAGE-6, GAGE-7, GAGE-8, GAGE-9), BAGE,
RAGE, LAGE-1, NAG, GnT-V, MUM-1, CDK4, tyrosinase, p53, MUC family,
HER2/neu, p21ras, RCAS1, .alpha.-fetoprotein, E-cadherin, 60
-catenin, .beta.-catenin and .gamma.-catenin, p120ctn, gp100
Pmel117, PRAME, NY-ESO-1, cdc27, adenomatous polyposis coli protein
(APC), fodrin, Connexin 37, Ig-idiotype, p15, gp75, GM2 and GD2
gangliosides, viral products such as human papilloma virus
proteins, Smad family of tumor antigens, lmp-1, NA, EBV-encoded
nuclear antigen (EBNA)-1, brain glycogen phosphorylase, SSX-1,
SSX-2 (HOM-MEL-40), SSX-1, SSX-4, SSX-5, SCP-1 CT-7, c-erbB-2,
CD19, CD20, CD22, CD30, CD33, CD37, CD56, CD70, CD74, CD138, AGS16,
MUC1, GPNMB, Ep-CAM, PD-L1, PD-L2, PMSA, bladder cancer antigens
such as ACTL8, ADAM22, ADAM23, ATAD2, ATAD2B, BIRC5, CASC5, CEP290,
CEP55, CTAGE5, DCAF12, DDX5, FAM133A, IL13RA2, IMP3, KIAA0100,
MAGEA11, MAGEA3, MAGEA6, MPHOSPH10, ODF2, ODF2L, OIP5, PBK, RQCD1,
SPAG1, SPAG4, SPAG9, TMEFF1, TTK, and prostate cancer antigens such
as PRAME, BIRC5, CEP55, ATAD2, ODF2, KIAA0100, SPAG9, GPATCH2,
ATAD2B, CEP290, SPAG1, ODF2L, CTAGE5, DDX5, DCAF12, IMP3. In some
embodiments, the antigens are human endogenous retroviral antigens.
Illustrative antigens can also include antigens from human
endogenous retroviruses which include, but are not limited to,
epitopes derived from at least a portion of Gag, at least a portion
of Tat, at least a portion of Rev, a least a portion of Nef, and at
least a portion of gp160.
[0096] Further, in some embodiments, the present vaccine protein
fusions (e.g., gp96 fusions) provide for an adjuvant effect that
further allows the immune system of a patient, when used in the
various methods described herein, to be activated against a disease
of interest.
[0097] Both prokaryotic and eukaryotic vectors can be used for
expression of the vaccine protein (e.g., gp96-Ig) and T cell
costimulatory fusion proteins in the methods provided herein.
Prokaryotic vectors include constructs based on E. coli sequences
(see, e.g., Makrides, Microbiol Rev 1996, 60:512-538). Non-limiting
examples of regulatory regions that can be used for expression in
E. coli include lac, trp, 1pp, phoA, recA, tac, T3, T7 and
.lamda.P.sub.L. Non-limiting examples of prokaryotic expression
vectors may include the Agt vector series such as .lamda.gt11
(Huynh et al., in "DNA Cloning Techniques, Vol. I: A Practical
Approach," 1984, (D. Glover, ed.), pp. 49-78, IRL Press, Oxford),
and the pET vector series (Studier et al., Methods Enzymol 1990,
185:60-89). Prokaryotic host-vector systems cannot perform much of
the post-translational processing of mammalian cells, however.
Thus, eukaryotic host-vector systems may be particularly
useful.
[0098] A variety of regulatory regions can be used for expression
of the vaccine protein (e.g., gp96-Ig) and T cell costimulatory
fusions in mammalian host cells. For example, the SV40 early and
late promoters, the cytomegalovirus (CMV) immediate early promoter,
and the Rous sarcoma virus long terminal repeat (RSV-LTR) promoter
can be used. Inducible promoters that may be useful in mammalian
cells include, without limitation, promoters associated with the
metallothionein II gene, mouse mammary tumor virus glucocorticoid
responsive long terminal repeats (MMTV-LTR), the n-interferon gene,
and the hsp70 gene (see, Williams et al., Cancer Res 1989,
49:2735-42; and Taylor et al., Mol Cell Biol 1990, 10:165-75). Heat
shock promoters or stress promoters also may be advantageous for
driving expression of the fusion proteins in recombinant host
cells.
[0099] In an embodiment, the present invention contemplates the use
of inducible promoters capable of effecting high level of
expression transiently in response to a cue. Illustrative inducible
expression control regions include those comprising an inducible
promoter that is stimulated with a cue such as a small molecule
chemical compound. Particular examples can be found, for example,
in U.S. Pat. Nos. 5,989,910, 5,935,934, 6,015,709, and 6,004,941,
each of which is incorporated herein by reference in its
entirety.
[0100] Animal regulatory regions that exhibit tissue specificity
and have been utilized in transgenic animals also can be used in
tumor cells of a particular tissue type: the elastase I gene
control region that is active in pancreatic acinar cells (Swift et
al., Cell 1984, 38:639-646; Ornitz et al., Cold Spring Harbor Symp
Quant Biol 1986, 50:399-409; and MacDonald, Hepatology 1987,
7:425-515); the insulin gene control region that is active in
pancreatic beta cells (Hanahan, Nature 1985, 315:115-122), the
immunoglobulin gene control region that is active in lymphoid cells
(Grosschedl et al., Cell 1984, 38:647-658; Adames et al., Nature
1985, 318:533-538; and Alexander et al., Mol Cell Biol 1987,
7:1436-1444), the mouse mammary tumor virus control region that is
active in testicular, breast, lymphoid and mast cells (Leder et
al., Cell 1986, 45:485-495), the albumin gene control region that
is active in liver (Pinkert et al., Genes Devel, 1987, 1:268-276),
the alpha-fetoprotein gene control region that is active in liver
(Krumlauf et al., Mol Cell Biol 1985, 5:1639-1648; and Hammer et
al., Science 1987, 235:53-58); the alpha 1-antitrypsin gene control
region that is active in liver (Kelsey et al., Genes Devel 1987,
1:161-171), the beta-globin gene control region that is active in
myeloid cells (Mogram et al., Nature 1985, 315:338-340; and Kollias
et al., Cell 1986, 46:89-94); the myelin basic protein gene control
region that is active in oligodendrocyte cells in the brain
(Readhead et al., Cell 1987, 48:703-712); the myosin light chain-2
gene control region that is active in skeletal muscle (Sani, Nature
1985, 314:283-286), and the gonadotropic releasing hormone gene
control region that is active in the hypothalamus (Mason et al.,
Science 1986, 234:1372-1378).
[0101] An expression vector also can include transcription enhancer
elements, such as those found in SV40 virus, Hepatitis B virus,
cytomegalovirus, immunoglobulin genes, metallothionein, and
.beta.-actin (see, Bittner et al., Meth Enzymol 1987, 153:516-544;
and Gorman, Curr Op Biotechnol 1990, 1:36-47). In addition, an
expression vector can contain sequences that permit maintenance and
replication of the vector in more than one type of host cell, or
integration of the vector into the host chromosome. Such sequences
include, without limitation, to replication origins, autonomously
replicating sequences (ARS), centromere DNA, and telomere DNA.
[0102] In addition, an expression vector can contain one or more
selectable or screenable marker genes for initially isolating,
identifying, or tracking host cells that contain DNA encoding
fusion proteins as described herein. For long term, high yield
production of gp96-Ig and T cell costimulatory fusion proteins,
stable expression in mammalian cells can be useful. A number of
selection systems can be used for mammalian cells. For example, the
Herpes simplex virus thymidine kinase (Wigler et al., Cell 1977,
11:223), hypoxanthine-guanine phosphoribosyltransferase (Szybalski
and Szybalski, Proc Natl Acad Sci USA 1962, 48:2026), and adenine
phosphoribosyltransferase (Lowy et al., Cell 1980, 22:817) genes
can be employed in tk.sup.-, hgprf.sup.-, or aprf.sup.- cells,
respectively. In addition, antimetabolite resistance can be used as
the basis of selection for dihydrofolate reductase (dhfr), which
confers resistance to methotrexate (Wigler et al., Proc Natl Acad
Sci USA 1980, 77:3567; O'Hare et al., Proc Natl Acad Sci USA 1981,
78:1527); gpt, which confers resistance to mycophenolic acid
(Mulligan and Berg, Proc Natl Acad Sci USA 1981, 78:2072); neomycin
phosphotransferase (neo), which confers resistance to the
aminoglycoside G-418 (Colberre-Garapin et al., J Mol Biol 1981,
150:1); and hygromycin phosphotransferase (hyg), which confers
resistance to hygromycin (Santerre et al., Gene 1984, 30:147).
Other selectable markers such as histidinol and Zeocin.TM. also can
be used.
[0103] Useful mammalian host cells include, without limitation,
cells derived from humans, monkeys, and rodents (see, for example,
Kriegler in "Gene Transfer and Expression: A Laboratory Manual,"
1990, New York, Freeman & Co.). These include monkey kidney
cell lines transformed by SV40 (e.g., COS-7, ATCC CRL 1651); human
embryonic kidney lines (e.g., 293, 293-EBNA, or 293 cells subcloned
for growth in suspension culture, Graham et al., J Gen Virol 1977,
36:59); baby hamster kidney cells (e.g., BHK, ATCC CCL 10); Chinese
hamster ovary-cells-DHFR (e.g., CHO, Urlaub and Chasin, Proc Natl
Acad Sci USA 1980, 77:4216); mouse sertoli cells (Mather, Biol
Reprod 1980, 23:243-251); mouse fibroblast cells (e.g., NIH-3T3),
monkey kidney cells (e.g., CV1 ATCC CCL 70); African green monkey
kidney cells. (e.g., VERO-76, ATCC CRL-1587); human cervical
carcinoma cells (e.g., HELA, ATCC CCL 2); canine kidney cells
(e.g., MDCK, ATCC CCL 34); buffalo rat liver cells (e.g., BRL 3A,
ATCC CRL 1442); human lung cells (e.g., W138, ATCC CCL 75); human
liver cells (e.g., Hep G2, HB 8065); and mouse mammary tumor cells
(e.g., MMT 060562, ATCC CCL51). Illustrative cancer cell types for
expressing the fusion proteins described herein include mouse
fibroblast cell line, NIH3T3, mouse Lewis lung carcinoma cell line,
LLC, mouse mastocytoma cell line, P815, mouse lymphoma cell line,
EL4 and its ovalbumin transfectant, E.G7, mouse melanoma cell line,
B16F10, mouse fibrosarcoma cell line, MC57, human small cell lung
carcinoma cell lines, SCLC#2 and SCLC#7, human lung adenocarcinoma
cell line, e.g., AD100, and human prostate cancer cell line, e.g.,
PC-3.
[0104] A number of viral-based expression systems also can be used
with mammalian cells to produce gp96-Ig and T cell costimulatory
fusion proteins. Vectors using DNA virus backbones have been
derived from simian virus 40 (SV40) (Hamer et al., Cell 1979,
17:725), adenovirus (Van Doren et al., Mol Cell Biol 1984, 4:1653),
adeno-associated virus (McLaughlin et al., J Virol 1988, 62:1963),
and bovine papillomas virus (Zinn et al., Proc Natl Acad Sci USA
1982, 79:4897). When an adenovirus is used as an expression vector,
the donor DNA sequence may be ligated to an adenovirus
transcription/translation control complex, e.g., the late promoter
and tripartite leader sequence. This fusion gene may then be
inserted in the adenovirus genome by in vitro or in vivo
recombination. Insertion in a non-essential region of the viral
genome (e.g., region E1 or E3) can result in a recombinant virus
that is viable and capable of expressing heterologous products in
infected hosts. (See, e.g., Logan and Shenk, Proc Natl Acad Sci USA
1984, 81:3655-3659).
[0105] Bovine papillomavirus (BPV) can infect many higher
vertebrates, including man, and its DNA replicates as an episome. A
number of shuttle vectors have been developed for recombinant gene
expression which exist as stable, multicopy (20-300 copies/cell)
extrachromosomal elements in mammalian cells. Typically, these
vectors contain a segment of BPV DNA (the entire genome or a 69%
transforming fragment), a promoter with a broad host range, a
polyadenylation signal, splice signals, a selectable marker, and
"poisonless" plasmid sequences that allow the vector to be
propagated in E. coli. Following construction and amplification in
bacteria, the expression gene constructs are transfected into
cultured mammalian cells by, for example, calcium phosphate
coprecipitation. For those host cells that do not manifest a
transformed phenotype, selection of transformants is achieved by
use of a dominant selectable marker, such as histidinol and G418
resistance.
[0106] Alternatively, the vaccinia 7.5K promoter can be used. (See,
e.g., Mackett et al., Proc Natl Acad Sci USA 1982, 79:7415-7419;
Mackett et al., J Virol 1984, 49:857-864; and Panicali et al., Proc
Natl Acad Sci USA 1982, 79:4927-4931.) In cases where a human host
cell is used, vectors based on the Epstein-Barr virus (EBV) origin
(OriP) and EBV nuclear antigen 1 (EBNA-1; a trans-acting
replication factor) can be used. Such vectors can be used with a
broad range of human host cells, e.g., EBO-pCD (Spickofsky et al.,
DNA Prot Eng Tech 1990, 2:14-18); pDR2 and .lamda.DR2 (available
from Clontech Laboratories).
[0107] Gp96-Ig and T cell costimulatory fusion proteins also can be
made with retrovirus-based expression systems. Retroviruses, such
as Moloney murine leukemia virus, can be used since most of the
viral gene sequence can be removed and replaced with exogenous
coding sequence while the missing viral functions can be supplied
in trans. In contrast to transfection, retroviruses can efficiently
infect and transfer genes to a wide range of cell types including,
for example, primary hematopoietic cells. Moreover, the host range
for infection by a retroviral vector can be manipulated by the
choice of envelope used for vector packaging.
[0108] For example, a retroviral vector can comprise a 5' long
terminal repeat (LTR), a 3' LTR, a packaging signal, a bacterial
origin of replication, and a selectable marker. The gp96-Ig fusion
protein coding sequence, for example, can be inserted into a
position between the 5' LTR and 3' LTR, such that transcription
from the 5' LTR promoter transcribes the cloned DNA. The 5' LTR
contains a promoter (e.g., an LTR promoter), an R region, a U5
region, and a primer binding site, in that order. Nucleotide
sequences of these LTR elements are well known in the art. A
heterologous promoter as well as multiple drug selection markers
also can be included in the expression vector to facilitate
selection of infected cells. See, McLauchlin et al., Prog Nucleic
Acid Res Mol Biol 1990, 38:91-135; Morgenstern et al., Nucleic Acid
Res 1990, 18:3587-3596; Choulika et al., J Virol 1996,
70:1792-1798; Boesen et al., Biotherapy 1994, 6:291-302; Salmons
and Gunzberg, Human Gene Ther 1993, 4:129-141; and Grossman and
Wilson, Curr Opin Genet Devel 1993, 3:110-114.
[0109] Any of the cloning and expression vectors described herein
may be synthesized and assembled from known DNA sequences using
techniques that are known in the art. The regulatory regions and
enhancer elements can be of a variety of origins, both natural and
synthetic. Some vectors and host cells may be obtained
commercially. Non-limiting examples of useful vectors are described
in Appendix 5 of Current Protocols in Molecular Biology, 1988, ed.
Ausubel et al., Greene Publish. Assoc. & Wiley Interscience,
which is incorporated herein by reference; and the catalogs of
commercial suppliers such as Clontech Laboratories, Stratagene
Inc., and Invitrogen, Inc.
Methods of Treating
[0110] An expression vector as provided herein can be incorporated
into a composition for administration to a subject (e.g., a
research animal or a mammal, such as a human, having a clinical
condition such as cancer or an infection). For example, an
expression vector can be administered to a subject for the
treatment of cancer or infection. Thus, this document provides
methods for treating clinical conditions such as cancer or
infection with the expression vectors provided herein. The
infection can be, for example, an acute infection or a chronic
infection. In some embodiments, the infection can be an infection
by hepatitis C virus, hepatitis B virus, human immunodeficiency
virus, or malaria. The methods can include administering to a
subject an expression vector, a cell containing the expression
vector, or a virus or virus-like particle containing the expression
vector, under conditions wherein the progression or a symptom of
the clinical condition in the subject is reduced in a therapeutic
manner.
[0111] In various embodiments, the present invention pertains to
cancers and/or tumors; for example, the treatment or prevention of
cancers and/or tumors. Cancers or tumors refer to an uncontrolled
growth of cells and/or abnormal increased cell survival and/or
inhibition of apoptosis which interferes with the normal
functioning of the bodily organs and systems. Included are benign
and malignant cancers, polyps, hyperplasia, as well as dormant
tumors or micrometastases. Also, included are cells having abnormal
proliferation that is not impeded by the immune system (e.g. virus
infected cells). The cancer may be a primary cancer or a metastatic
cancer. The primary cancer may be an area of cancer cells at an
originating site that becomes clinically detectable, and may be a
primary tumor. In contrast, the metastatic cancer may be the spread
of a disease from one organ or part to another non-adjacent organ
or part. The metastatic cancer may be caused by a cancer cell that
acquires the ability to penetrate and infiltrate surrounding normal
tissues in a local area, forming a new tumor, which may be a local
metastasis. The cancer may also be caused by a cancer cell that
acquires the ability to penetrate the walls of lymphatic and/or
blood vessels, after which the cancer cell is able to circulate
through the bloodstream (thereby being a circulating tumor cell) to
other sites and tissues in the body. The cancer may be due to a
process such as lymphatic or hematogeneous spread. The cancer may
also be caused by a tumor cell that comes to rest at another site,
re-penetrates through the vessel or walls, continues to multiply,
and eventually forms another clinically detectable tumor. The
cancer may be this new tumor, which may be a metastatic (or
secondary) tumor.
[0112] The cancer may be caused by tumor cells that have
metastasized, which may be a secondary or metastatic tumor. The
cells of the tumor may be like those in the original tumor. As an
example, if a breast cancer or colon cancer metastasizes to the
liver, the secondary tumor, while present in the liver, is made up
of abnormal breast or colon cells, not of abnormal liver cells. The
tumor in the liver may thus be a metastatic breast cancer or a
metastatic colon cancer, not liver cancer.
[0113] The cancer may have an origin from any tissue. The cancer
may originate from melanoma, colon, breast, or prostate, and thus
may be made up of cells that were originally skin, colon, breast,
or prostate, respectively. The cancer may also be a hematological
malignancy, which may be lymphoma. The cancer may invade a tissue
such as liver, lung, bladder, or intestinal.
[0114] Illustrative cancers that may be treated include, but are
not limited to, carcinomas, e.g. various subtypes, including, for
example, adenocarcinoma, basal cell carcinoma, squamous cell
carcinoma, and transitional cell carcinoma), sarcomas (including,
for example, bone and soft tissue), leukemias (including, for
example, acute myeloid, acute lymphoblastic, chronic myeloid,
chronic lymphocytic, and hairy cell), lymphomas and myelomas
(including, for example, Hodgkin and non-Hodgkin lymphomas, light
chain, non-secretory, MGUS, and plasmacytomas), and central nervous
system cancers (including, for example, brain (e.g. gliomas (e.g.
astrocytoma, oligodendroglioma, and ependymoma), meningioma,
pituitary adenoma, and neuromas, and spinal cord tumors (e.g.
meningiomas and neurofibroma).
[0115] Representative cancers and/or tumors of the present
invention include, but are not limited to, a basal cell carcinoma,
biliary tract cancer; bladder cancer; bone cancer; brain and
central nervous system cancer; breast cancer; cancer of the
peritoneum; cervical cancer; choriocarcinoma; colon and rectum
cancer; connective tissue cancer; cancer of the digestive system;
endometrial cancer; esophageal cancer; eye cancer; cancer of the
head and neck; gastric cancer (including gastrointestinal cancer);
glioblastoma; hepatic carcinoma; hepatoma; intra-epithelial
neoplasm; kidney or renal cancer; larynx cancer; leukemia; liver
cancer; lung cancer (e.g., small-cell lung cancer, non-small cell
lung cancer, adenocarcinoma of the lung, and squamous carcinoma of
the lung); melanoma; myeloma; neuroblastoma; oral cavity cancer
(lip, tongue, mouth, and pharynx); ovarian cancer; pancreatic
cancer; prostate cancer; retinoblastoma; rhabdomyosarcoma; rectal
cancer; cancer of the respiratory system; salivary gland carcinoma;
sarcoma; skin cancer; squamous cell cancer; stomach cancer;
testicular cancer; thyroid cancer; uterine or endometrial cancer;
cancer of the urinary system; vulval cancer; lymphoma including
Hodgkin's and non-Hodgkin's lymphoma, as well as B-cell lymphoma
(including low grade/follicular non-Hodgkin's lymphoma (NHL); small
lymphocytic (SL) NHL; intermediate grade/follicular NHL;
intermediate grade diffuse NHL; high grade immunoblastic NHL; high
grade lymphoblastic NHL; high grade small non-cleaved cell NHL;
bulky disease NHL; mantle cell lymphoma; AIDS-related lymphoma; and
Waldenstrom's Macroglobulinemia; chronic lymphocytic leukemia
(CLL); acute lymphoblastic leukemia (ALL); Hairy cell leukemia;
chronic myeloblastic leukemia; as well as other carcinomas and
sarcomas; and post-transplant lymphoproliferative disorder (PTLD),
as well as abnormal vascular proliferation associated with
phakomatoses, edema (such as that associated with brain tumors),
and Meigs' syndrome.
[0116] In some aspects, the present fusions are used to eliminate
intracellular pathogens. In some aspects, the present fusions are
used to treat one or more infections. In some embodiments, the
present fusion proteins are used in methods of treating viral
infections (including, for example, HIV and HCV), parasitic
infections (including, for example, malaria), and bacterial
infections. In various embodiments, the infections induce
immunosuppression. For example, HIV infections often result in
immunosuppression in the infected subjects. Accordingly, as
described elsewhere herein, the treatment of such infections may
involve, in various embodiments, modulating the immune system with
the present fusion proteins to favor immune stimulation over immune
inhibition. Alternatively, the present invention provides methods
for treating infections that induce immunoactivation. For example,
intestinal helminth infections have been associated with chronic
immune activation. In these embodiments, the treatment of such
infections may involve modulating the immune system with the
present fusion proteins to favor immune inhibition over immune
stimulation.
[0117] In various embodiments, the present invention provides
methods of treating viral infections including, without limitation,
acute or chronic viral infections, for example, of the respiratory
tract, of papilloma virus infections, of herpes simplex virus (HSV)
infection, of human immunodeficiency virus (HIV) infection, and of
viral infection of internal organs such as infection with hepatitis
viruses. In some embodiments, the viral infection is caused by a
virus of family Flaviviridae. In some embodiments, the virus of
family Flaviviridae is selected from Yellow Fever Virus, West Nile
virus, Dengue virus, Japanese Encephalitis Virus, St. Louis
Encephalitis Virus, and Hepatitis C Virus. In other embodiments,
the viral infection is caused by a virus of family Picornaviridae,
e.g., poliovirus, rhinovirus, coxsackievirus. In other embodiments,
the viral infection is caused by a member of Orthomyxoviridae,
e.g., an influenza virus. In other embodiments, the viral infection
is caused by a member of Retroviridae, e.g., a lentivirus. In other
embodiments, the viral infection is caused by a member of
Paramyxoviridae, e.g., respiratory syncytial virus, a human
parainfluenza virus, rubulavirus (e.g., mumps virus), measles
virus, and human metapneumovirus. In other embodiments, the viral
infection is caused by a member of Bunyaviridae, e.g., hantavirus.
In other embodiments, the viral infection is caused by a member of
Reoviridae, e.g., a rotavirus.
[0118] In various embodiments, the present invention provides
methods of treating parasitic infections such as protozoan or
helminths infections. In some embodiments, the parasitic infection
is by a protozoan parasite. In some embodiments, the oritiziab
parasite is selected from intestinal protozoa, tissue protozoa, or
blood protozoa. Illustrative protozoan parasites include, but are
not limited to, Entamoeba hystolytica, Giardia lamblia,
Cryptosporidium muris, Trypanosomatida gambiense, Trypanosomatida
rhodesiense, Trypanosomatida crusi, Leishmania mexicana, Leishmania
braziliensis, Leishmania tropica, Leishmania donovani, Toxoplasma
gondii, Plasmodium vivax, Plasmodium ovale, Plasmodium malariae,
Plasmodium falciparum, Trichomonas vaginalis, and Histomonas
meleagridis. In some embodiments, the parasitic infection is by a
helminthic parasite such as nematodes (e.g., Adenophorea). In some
embodiments, the parasite is selected from Secementea (e.g.,
Trichuris trichiura, Ascaris lumbricoides, Enterobius vermicularis,
Ancylostoma duodenale, Necator americanus, Strongyloides
stercoralis, Wuchereria bancrofti, Dracunculus medinensis). In some
embodiments, the parasite is selected from trematodes (e.g. blood
flukes, liver flukes, intestinal flukes, and lung flukes). In some
embodiments, the parasite is selected from: Schistosoma mansoni,
Schistosoma haematobium, Schistosoma japonicum, Fasciola hepatica,
Fasciola gigantica, Heterophyes heterophyes, Paragonimus
westermani. In some embodiments, the parasite is selected from
cestodes (e.g., Taenia solium, Taenia saginata, Hymenolepis nana,
Echinococcus granulosus).
[0119] In various embodiments, the present invention provides
methods of treating bacterial infections. In various embodiments,
the bacterial infection is by a gram-positive bacteria,
gram-negative bacteria, aerobic and/or anaerobic bacteria. In
various embodiments, the bacteria is selected from, but not limited
to, Staphylococcus, Lactobacillus, Streptococcus, Sarcina,
Escherichia, Enterobacter, Klebsiella, Pseudomonas, Acinetobacter,
Mycobacterium, Proteus, Campylobacter, Citrobacter, Nisseria,
Baccillus, Bacteroides, Peptococcus, Clostridium, Salmonella,
Shigella, Serratia, Haemophilus, Brucella and other organisms. In
some embodiments, the bacteria is selected from, but not limited
to, Pseudomonas aeruginosa, Pseudomonas fluorescens, Pseudomonas
acidovorans, Pseudomonas alcaligenes, Pseudomonas pufida,
Stenotrophomonas maltophilia, Burkholderia cepacia, Aeromonas
hydrophilia, Escherichia coli, Citrobacter freundii, Salmonella
typhimurium, Salmonella typhi, Salmonella paratyphi, Salmonella
enteritidis, Shigella dysenteriae, Shigella flexneri, Shigella
sonnei, Enterobacter cloacae, Enterobacter aerogenes, Klebsiella
pneumoniae, Klebsiella oxytoca, Serratia marcescens, Francisella
tularensis, Morganella morganii, Proteus mirabilis, Proteus
vulgaris, Providencia alcalifaciens, Providencia rettgeri,
Providencia stuartii, Acinetobacter baumannii, Acinetobacter
calcoaceficus, Acinetobacter haemolyticus, Yersinia enterocolitica,
Yersinia pestis, Yersinia pseudotuberculosis, Yersinia intermedia,
Bordetella pertussis, Bordetella parapertussis, Bordetella
bronchiseptica, Haemophilus influenzae, Haemophilus parainfluenzae,
Haemophilus haemolyticus, Haemophilus parahaemolyticus, Haemophilus
ducreyi, Pasteurella multocida, Pasteurella haemolytica,
Branhamella catarrhalis, Helicobacter pylori, Campylobacter fetus,
Campylobacter jejuni, Campylobacter coli, Borrelia burgdorferi,
Vibrio cholerae, Vibrio parahaemolyticus, Legionella pneumophila,
Listeria monocytogenes, Neisseria gonorrhoeae, Neisseria
meningitidis, Kingella, Moraxella, Gardnerella vaginalis,
Bacteroides fragilis, Bacteroides distasonis, Bacteroides 3452A
homology group, Bacteroides vulgatus, Bacteroides ovalus,
Bacteroides thetaiotaomicron, Bacteroides uniformis, Bacteroides
eggerthii, Bacteroides splanchnicus, Clostridium difficile,
Mycobacterium tuberculosis, Mycobacterium avium, Mycobacterium
intracellulare, Mycobacterium leprae, Corynebacterium diphtheriae,
Corynebacterium ulcerans, Streptococcus pneumoniae, Streptococcus
agalactiae, Streptococcus pyogenes, Enterococcus faecalis,
Enterococcus faecium, Staphylococcus aureus, Staphylococcus
epidermidis, Staphylococcus saprophyticus, Staphylococcus
intermedius, Staphylococcus hyicus subsp. hyicus, Staphylococcus
haemolyticus, Staphylococcus hominis, or Staphylococcus
saccharolyticus. The expression vector(s), cells, or particles to
be administered can be admixed, encapsulated, conjugated or
otherwise associated with other molecules, molecular structures, or
mixtures of compounds such as, for example, liposomes, receptor or
cell targeted molecules, or oral, topical or other formulations for
assisting in uptake, distribution and/or absorption. In some cases,
an expression vector can be contained within a cell that is
administered to a subject, or contained within a virus or
virus-like particle. The vector, cell, or particle to be
administered can be in combination with a pharmaceutically
acceptable carrier.
[0120] This document therefore also provides compositions
containing a vector or a tumor cell or virus particle containing a
vector encoding a secreted gp96-Ig fusion polypeptide and a T cell
costimulatory fusion polypeptide as described herein, in
combination with a physiologically and pharmaceutically acceptable
carrier. The physiologically and pharmaceutically acceptable
carrier can be include any of the well-known components useful for
immunization. The carrier can facilitate or enhance an immune
response to an antigen administered in a vaccine. The cell
formulations can contain buffers to maintain a preferred pH range,
salts or other components that present an antigen to an individual
in a composition that stimulates an immune response to the antigen.
The physiologically acceptable carrier also can contain one or more
adjuvants that enhance the immune response to an antigen.
Pharmaceutically acceptable carriers include, for example,
pharmaceutically acceptable solvents, suspending agents, or any
other pharmacologically inert vehicles for delivering compounds to
a subject. Pharmaceutically acceptable carriers can be liquid or
solid, and can be selected with the planned manner of
administration in mind so as to provide for the desired bulk,
consistency, and other pertinent transport and chemical properties,
when combined with one or more therapeutic compounds and any other
components of a given pharmaceutical composition. Typical
pharmaceutically acceptable carriers include, without limitation:
water, saline solution, binding agents (e.g., polyvinylpyrrolidone
or hydroxypropyl methylcellulose); fillers (e.g., lactose or
dextrose and other sugars, gelatin, or calcium sulfate), lubricants
(e.g., starch, polyethylene glycol, or sodium acetate),
disintegrates (e.g., starch or sodium starch glycolate), and
wetting agents (e.g., sodium lauryl sulfate). Compositions can be
formulated for subcutaneous, intramuscular, or intradermal
administration, or in any manner acceptable for immunization.
[0121] An adjuvant refers to a substance which, when added to an
immunogenic agent such as a tumor cell expressing secreted vaccine
protein (e.g., gp96-Ig) and T cell costimulatory fusion
polypeptides, nonspecifically enhances or potentiates an immune
response to the agent in the recipient host upon exposure to the
mixture. Adjuvants can include, for example, oil-in-water
emulsions, water-in oil emulsions, alum (aluminum salts), liposomes
and microparticles, such as, polysytrene, starch, polyphosphazene
and polylactide/polyglycosides.
[0122] Adjuvants can also include, for example, squalene mixtures
(SAF-I), muramyl peptide, saponin derivatives, mycobacterium cell
wall preparations, monophosphoryl lipid A, mycolic acid
derivatives, nonionic block copolymer surfactants, Quil A, cholera
toxin B subunit, polyphosphazene and derivatives, and
immunostimulating complexes (ISCOMs) such as those described by
Takahashi et al., Nature 1990, 344:873-875. For veterinary use and
for production of antibodies in animals, mitogenic components of
Freund's adjuvant (both complete and incomplete) can be used. In
humans, Incomplete Freund's Adjuvant (IFA) is a useful adjuvant.
Various appropriate adjuvants are well known in the art (see, for
example, Warren and Chedid, CRC Critical Reviews in Immunology
1988, 8:83; and Allison and Byars, in Vaccines: New Approaches to
Immunological Problems, 1992, Ellis, ed., Butterworth-Heinemann,
Boston). Additional adjuvants include, for example, bacille
Calmett-Guerin (BCG), DETOX (containing cell wall skeleton of
Mycobacterium phlei (CWS) and monophosphoryl lipid A from
Salmonella minnesota (MPL)), and the like (see, for example, Hoover
et al., J Clin Oncol 1993, 11:390; and Woodlock et al., J
Immunother 1999, 22:251-259).
[0123] In some embodiments, a vector can be administered to a
subject one or more times (e.g., once, twice, two to four times,
three to five times, five to eight times, six to ten times, eight
to 12 times, or more than 12 times). A vector as provided herein
can be administered one or more times per day, one or more times
per week, every other week, one or more times per month, once every
two to three months, once every three to six months, or once every
six to 12 months. A vector can be administered over any suitable
period of time, such as a period from about 1 day to about 12
months. In some embodiments, for example, the period of
administration can be from about 1 day to 90 days; from about 1 day
to 60 days; from about 1 day to 30 days; from about 1 day to 20
days; from about 1 day to 10 days; from about 1 day to 7 days. In
some embodiments, the period of administration can be from about 1
week to 50 weeks; from about 1 week to 50 weeks; from about 1 week
to 40 weeks; from about 1 week to 30 weeks; from about 1 week to 24
weeks; from about 1 week to 20 weeks; from about 1 week to 16
weeks; from about 1 week to 12 weeks; from about 1 week to 8 weeks;
from about 1 week to 4 weeks; from about 1 week to 3 weeks; from
about 1 week to 2 weeks; from about 2 weeks to 3 weeks; from about
2 weeks to 4 weeks; from about 2 weeks to 6 weeks; from about 2
weeks to 8 weeks; from about 3 weeks to 8 weeks; from about 3 weeks
to 12 weeks; or from about 4 weeks to 20 weeks.
[0124] In some embodiments, after an initial dose (sometimes
referred to as a "priming" dose) of a vector has been administered
and a maximal antigen-specific immune response has been achieved,
one or more boosting doses of a vector as provided herein can be
administered. For example, a boosting dose can be administered
about 10 to 30 days, about 15 to 35 days, about 20 to 40 days,
about 25 to 45 days, or about 30 to 50 days after a priming
dose.
[0125] In some embodiments, the methods provided herein can be used
for controlling solid tumor growth (e.g., breast, prostate,
melanoma, renal, colon, or cervical tumor growth) and/or
metastasis. The methods can include administering an effective
amount of an expression vector as described herein to a subject in
need thereof. In some embodiments, the subject is a mammal (e g., a
human).
[0126] The vectors and methods provided herein can be useful for
stimulating an immune response against a tumor. Such immune
response is useful in treating or alleviating a sign or symptom
associated with the tumor. As used herein, by "treating" is meant
reducing, preventing, and/or reversing the symptoms in the
individual to which a vector as described herein has been
administered, as compared to the symptoms of an individual not
being treated. A practitioner will appreciate that the methods
described herein are to be used in concomitance with continuous
clinical evaluations by a skilled practitioner (physician or
veterinarian) to determine subsequent therapy. Such evaluations
will aid and inform in evaluating whether to increase, reduce, or
continue a particular treatment dose, mode of administration,
etc.
[0127] The methods provided herein can thus be used to treat a
tumor, including, for example, a cancer. The methods can be used,
for example, to inhibit the growth of a tumor by preventing further
tumor growth, by slowing tumor growth, or by causing tumor
regression. Thus, the methods can be used, for example, to treat a
cancer such as a lung cancer. It will be understood that the
subject to which a compound is administered need not suffer from a
specific traumatic state. Indeed, the vectors described herein may
be administered prophylactically, prior to development of symptoms
(e.g., a patient in remission from cancer). The terms "therapeutic"
and "therapeutically," and permutations of these terms, are used to
encompass therapeutic, palliative, and prophylactic uses. Thus, as
used herein, by "treating or alleviating the symptoms" is meant
reducing, preventing, and/or reversing the symptoms of the
individual to which a therapeutically effective amount of a
composition has been administered, as compared to the symptoms of
an individual receiving no such administration.
[0128] As used herein, the terms "effective amount" and
"therapeutically effective amount" refer to an amount sufficient to
provide the desired therapeutic (e.g., anti-cancer, anti-tumor, or
anti-infection) effect in a subject (e.g., a human diagnosed as
having cancer or an infection). Anti-tumor and anti-cancer effects
include, without limitation, modulation of tumor growth (e.g.,
tumor growth delay), tumor size, or metastasis, the reduction of
toxicity and side effects associated with a particular anti-cancer
agent, the amelioration or minimization of the clinical impairment
or symptoms of cancer, extending the survival of the subject beyond
that which would otherwise be expected in the absence of such
treatment, and the prevention of tumor growth in an animal lacking
tumor formation prior to administration, i.e., prophylactic
administration. In some embodiments, administration of an effective
amount of a vector or a composition, cell, or virus particle
containing the vector can increase the activation or proliferation
of tumor antigen specific T cells in a subject. For example, the
activation or proliferation of tumor antigen specific T cells in
the subject can be is increased by at least 10 percent (e.g., at
least 25 percent, at least 50 percent, or at least 75 percent) as
compared to the level of activation or proliferation of tumor
antigen specific T cells in the subject prior to the
administration.
[0129] Anti-infection effects include, for example, a reduction in
the number of infective agents (e.g., viruses or bacteria). When
the clinical condition in the subject to be treated is an
infection, administration of a vector as provided herein can
stimulate the activation or proliferation of pathogenic antigen
specific T cells in the subject. For example, administration of the
vector can lead to activation of antigen-specific T cells in the
subject to a level great than that achieved by 96-Ig vaccination
alone.
[0130] One of skill will appreciate that an effective amount of a
vector may be lowered or increased by fine tuning and/or by
administering more than one dose (e.g., by concomitant
administration of two different genetically modified tumor cells
containing the vector), or by administering a vector with another
agent (e.g., an antagonist of PD-1) to enhance the therapeutic
effect (e.g., synergistically). This document therefore provides a
method for tailoring the administration/treatment to the particular
exigencies specific to a given mammal. Therapeutically effective
amounts can be determined by, for example, starting at relatively
low amounts and using step-wise increments with concurrent
evaluation of beneficial effects. The methods provided herein thus
can be used alone or in combination with other well-known tumor
therapies, to treat a patient having a tumor. One skilled in the
art will readily understand advantageous uses of the vectors and
methods provided herein, for example, in prolonging the life
expectancy of a cancer patient and/or improving the quality of life
of a cancer patient (e.g., a lung cancer patient).
Combination Therapies and Conjugation
[0131] In some embodiments, the invention provides for methods that
further comprise administering an additional agent to a subject. In
some embodiments, the invention pertains to co-administration
and/or co-formulation.
[0132] In some embodiments, administration of vaccine protein
(e.g., gp96-Ig) and one or more costimulatory molecules act
synergistically when co-administered with another agent and is
administered at doses that are lower than the doses commonly
employed when such agents are used as monotherapy.
[0133] In some embodiments, inclusive of, without limitation,
cancer applications, the present invention pertains to
chemotherapeutic agents as additional agents. Examples of
chemotherapeutic agents include, but are not limited to, alkylating
agents such as thiotepa and CYTOXAN cyclosphosphamide; alkyl
sulfonates such as busulfan, improsulfan and piposulfan; aziridines
such as benzodopa, carboquone, meturedopa, and uredopa;
ethylenimines and methylamelamines including altretamine,
triethylenemelamine, trietylenephosphoramide,
triethiylenethiophosphoramide and trimethylolomelamine; acetogenins
(e.g., bullatacin and bullatacinone); a camptothecin (including the
synthetic analogue topotecan); bryostatin; cally statin; CC-1065
(including its adozelesin, carzelesin and bizelesin synthetic
analogues); cryptophycins (e.g., cryptophycin 1 and cryptophycin
8); dolastatin; duocarmycin (including the synthetic analogues,
KW-2189 and CB 1-TM1); eleutherobin; pancratistatin; a
sarcodictyin; spongistatin; nitrogen mustards such as chlorambucil,
chlornaphazine, cholophosphamide, estramustine, ifosfamide,
mechlorethamine, mechlorethamine oxide hydrochloride, melphalan,
novembichin, phenesterine, prednimustine, trofosfamide, uracil
mustard; nitrosureas such as carmustine, chlorozotocin,
fotemustine, lomustine, nimustine, and ranimnustine; antibiotics
such as the enediyne antibiotics (e.g., calicheamicin, especially
calicheamicin gammall and calicheamicin omegall (see, e.g., Agnew,
Chem. Intl. Ed. Engl., 33: 183-186 (1994)); dynemicin, including
dynemicin A; bisphosphonates, such as clodronate; an esperamicin;
as well as neocarzinostatin chromophore and related chromoprotein
enediyne antibiotic chromophores), aclacinomy sins, actinomycin,
authramycin, azaserine, bleomycins, cactinomycin, carabicin,
caminomycin, carzinophilin, chromomycinis, dactinomycin,
daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, ADRIAMYCIN
doxorubicin (including morpholino-doxorubicin,
cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and deoxy
doxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin,
mitomycins such as mitomycin C, mycophenolic acid, nogalamycin,
olivomycins, peplomycin, potfiromycin, puromycin, quelamycin,
rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex,
zinostatin, zorubicin; anti-metabolites such as methotrexate and
5-fluorouracil (5-FU); folic acid analogues such as denopterin,
methotrexate, pteropterin, trimetrexate; purine analogs such as
fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine
analogs such as ancitabine, azacitidine, 6-azauridine, carmofur,
cytarabine, dideoxyuridine, doxifluridine, enocitabine,
floxuridine; androgens such as calusterone, dromostanolone
propionate, epitiostanol, mepitiostane, testolactone; anti-adrenals
such as minoglutethimide, mitotane, trilostane; folic acid
replenisher such as frolinic acid; aceglatone; aldophosphamide
glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil;
bisantrene; edatraxate; demecolcine; diaziquone; elformithine;
elliptinium acetate; an epothilone; etoglucid; gallium nitrate;
hydroxyurea; lentinan; lonidainine; maytansinoids such as
maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol;
nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone;
podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK
polysaccharide complex (JHS Natural Products, Eugene, Oreg.);
razoxane; rhizoxin; sizofuran; spirogermanium; tenuazonic acid;
triaziquone; 2,2',2''-trichlorotriethylamine; trichothecenes (e.g.,
T-2 toxin, verracurin A, roridin A and anguidine); urethan;
vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol;
pipobroman; gacytosine; arabinoside ("Ara-C"); cyclophosphamide;
thiotepa; taxoids, e.g., TAXOL paclitaxel (Bristol-Myers Squibb
Oncology, Princeton, N.J.), ABRAXANE Cremophor-free,
albumin-engineered nanoparticle formulation of paclitaxel (American
Pharmaceutical Partners, Schaumberg, Ill.), and TAXOTERE doxetaxel
(Rhone-Poulenc Rorer, Antony, France); chloranbucil; GEMZAR
gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum
analogs such as cisplatin, oxaliplatin and carboplatin;
vinblastine; platinum; etoposide (VP-16); ifosfamide; mitoxantrone;
vincristine; NAVELBINE. vinorelbine; novantrone; teniposide;
edatrexate; daunomycin; aminopterin; xeloda; ibandronate;
irinotecan (Camptosar, CPT-11) (including the treatment regimen of
irinotecan with 5-FU and leucovorin); topoisomerase inhibitor RFS
2000; difluoromethylornithine (DMFO); retinoids such as retinoic
acid; capecitabine; combretastatin; leucovorin (LV); oxaliplatin,
including the oxaliplatin treatment regimen (FOLFOX); lapatinib
(TYKERB); inhibitors of PKC-.alpha., Raf, H-Ras, EGFR (e.g.,
erlotinib (Tarceva)) and VEGF-A that reduce cell proliferation and
pharmaceutically acceptable salts, acids or derivatives of any of
the above. In addition, the methods of treatment can further
include the use of radiation. In addition, the methods of treatment
can further include the use of photodynamic therapy.
[0134] In some embodiments, inclusive of, without limitation,
infectious disease applications, the present invention pertains to
anti-infectives as additional agents. In some embodiments, the
anti-infective is an anti-viral agent including, but not limited
to, Abacavir, Acyclovir, Adefovir, Amprenavir, Atazanavir,
Cidofovir, Darunavir, Delavirdine, Didanosine, Docosanol,
Efavirenz, Elvitegravir, Emtricitabine, Enfuvirtide, Etravirine,
Famciclovir, and Foscarnet. In some embodiments, the anti-infective
is an anti-bacterial agent including, but not limited to,
cephalosporin antibiotics (cephalexin, cefuroxime, cefadroxil,
cefazolin, cephalothin, cefaclor, cefamandole, cefoxitin,
cefprozil, and ceftobiprole); fluoroquinolone antibiotics (cipro,
Levaquin, floxin, tequin, avelox, and norflox); tetracycline
antibiotics (tetracycline, minocycline, oxytetracycline, and
doxycycline); penicillin antibiotics (amoxicillin, ampicillin,
penicillin V, dicloxacillin, carbenicillin, vancomycin, and
methicillin); monobactam antibiotics (aztreonam); and carbapenem
antibiotics (ertapenem, doripenem, imipenem/cilastatin, and
meropenem). In some embodiments, the anti-infectives include
anti-malarial agents (e.g., chloroquine, quinine, mefloquine,
primaquine, doxycycline, artemether/lumefantrine,
atovaquone/proguanil and sulfadoxine/pyrimethamine), metronidazole,
tinidazole, ivermectin, pyrantel pamoate, and albendazole.
[0135] Other additional agents are described elsewhere herein,
including the blocking antibodies targeted to an immune
"checkpoint" molecules.
Subjects and/or Animals
[0136] The methods described herein are intended for use with any
subject that may experience the benefits of these methods. Thus,
"subjects," "patients," and "individuals" (used interchangeably)
include humans as well as non-human subjects, particularly
domesticated animals.
[0137] In some embodiments, the subject and/or animal is a mammal,
e g., a human, mouse, rat, guinea pig, dog, cat, horse, cow, pig,
rabbit, sheep, or non-human primate, such as a monkey, chimpanzee,
or baboon. In other embodiments, the subject and/or animal is a
non-mammal, such, for example, a zebrafish. In some embodiments,
the subject and/or animal may comprise fluorescently-tagged cells
(with e.g. GFP). In some embodiments, the subject and/or animal is
a transgenic animal comprising a fluorescent cell.
[0138] In some embodiments, the subject and/or animal is a human In
some embodiments, the human is a pediatric human In other
embodiments, the human is an adult human. In other embodiments, the
human is a geriatric human In other embodiments, the human may be
referred to as a patient.
[0139] In certain embodiments, the human has an age in a range of
from about 0 months to about 6 months old, from about 6 to about 12
months old, from about 6 to about 18 months old, from about 18 to
about 36 months old, from about 1 to about 5 years old, from about
5 to about 10 years old, from about 10 to about 15 years old, from
about 15 to about 20 years old, from about 20 to about 25 years
old, from about 25 to about 30 years old, from about 30 to about 35
years old, from about 35 to about 40 years old, from about 40 to
about 45 years old, from about 45 to about 50 years old, from about
50 to about 55 years old, from about 55 to about 60 years old, from
about 60 to about 65 years old, from about 65 to about 70 years
old, from about 70 to about 75 years old, from about 75 to about 80
years old, from about 80 to about 85 years old, from about 85 to
about 90 years old, from about 90 to about 95 years old or from
about 95 to about 100 years old.
[0140] In other embodiments, the subject is a non-human animal, and
therefore the invention pertains to veterinary use. In a specific
embodiment, the non-human animal is a household pet. In another
specific embodiment, the non-human animal is a livestock animal In
certain embodiments, the subject is a human cancer patient that
cannot receive chemotherapy, e.g. the patient is unresponsive to
chemotherapy or too ill to have a suitable therapeutic window for
chemotherapy (e.g. experiencing too many dose- or regimen-limiting
side effects). In certain embodiments, the subject is a human
cancer patient having advanced and/or metastatic disease.
[0141] As used herein, an "allogeneic cell" refers to a cell that
is not derived from the individual to which the cell is to be
administered, that is, has a different genetic constitution than
the individual. An allogeneic cell is generally obtained from the
same species as the individual to which the cell is to be
administered. For example, the allogeneic cell can be a human cell,
as disclosed herein, for administering to a human patient such as a
cancer patient. As used herein, an "allogeneic tumor cell" refers
to a tumor cell that is not derived from the individual to which
the allogeneic cell is to be administered. Generally, the
allogeneic tumor cell expresses one or more tumor antigens that can
stimulate an immune response against a tumor in an individual to
which the cell is to be administered. As used herein, an
"allogeneic cancer cell," for example, a lung cancer cell, refers
to a cancer cell that is not derived from the individual to which
the allogeneic cell is to be administered.
[0142] As used herein, a "genetically modified cell" refers to a
cell that has been genetically modified to express an exogenous
nucleic acid, for example, by transfection or transduction.
[0143] Technical and scientific terms used herein have the meaning
commonly understood by one of skill in the art to which the present
invention pertains, unless otherwise defined.
[0144] As used herein, the singular forms "a," "an" and "the"
specifically also encompass the plural forms of the terms to which
they refer, unless the content clearly dictates otherwise. As used
herein, unless specifically indicated otherwise, the word "or" is
used in the "inclusive" sense of "and/or" and not the "exclusive"
sense of either/or." In the specification and the appended claims,
the singular forms include plural referents unless the context
clearly dictates otherwise.
[0145] The term "about" is used herein to mean approximately, in
the region of, roughly, or around. When the term "about" is used in
conjunction with a numerical range, it modifies that range by
extending the boundaries above and below the numerical values set
forth. In general, the term "about" is used herein to modify a
numerical value above and below the stated value by a variance of
20%. As used in this specification, whether in a transitional
phrase or in the body of the claim, the terms "comprise (s)" and
"comprising" are to be interpreted as having an open-ended meaning.
That is, the terms are to be interpreted synonymously with the
phrases "having at least" or "including at least". When used in the
context of a process, the term "comprising" means that the process
includes at least the recited steps, but may include additional
steps. When used in the context of a compound or composition, the
term "comprising" means that the compound or composition includes
at least the recited features or components, but may also include
additional features or components.
[0146] The invention will be further described in the following
examples, which do not limit the scope of the invention described
in the claims.
EXAMPLES
Example 1
Vector Engineered Immunotherapy Incorporating gp96-Ig and T Cell
Costimulatory Fusion Proteins Elicits a Superior Antigen-Specific
CD8+ T Cell Response
[0147] Secretable heat-shock protein gp96-Ig based allogeneic
cellular vaccines can achieve high frequency polyclonal CD8+ T cell
responses to femtomolar concentrations of tumor antigens through
antigen cross-priming in vivo. Multiple immunosuppressive
mechanisms evolved by established tumors can dampen the activity of
this vaccine approach. As described below, a systematic comparison
of PD-1, PD-L1, CTLA-4, and LAG-3 blocking antibodies in mouse
models of long-established B16-F10 melanoma demonstrated a superior
combination between gp96-Ig vaccination and PD-1 blockade as
compared to other checkpoints. Triple combinations of gp96-Ig
vaccination, PD-1 blockade, and T cell costimulation using OX40,
ICOS, or 4-1BB agonists provided a synergistic anti-tumor
benefit.
[0148] A 96-Ig expression vector was re-engineered to
simultaneously co-express ICOSL-Ig, 4-1BBL-Ig, or OX40L-Ig, thus
providing a costimulatory benefit without the need for additional
antibody therapy. As described below, co-secretion of gp96-Ig and
these costimulatory fusion proteins in allogeneic cell lines
resulted in enhanced activation of antigen-specific CD8+ T cells.
Thus, combination immunotherapy can be achieved by vector
re-engineering, obviating the need for vaccine/antibody/fusion
protein regimens, and importantly may limit both cost of therapy
and the risk of systemic toxicity.
Example 2
Vaccine+Costimulator Vector Re-Engineering
[0149] A vector re-engineering strategy was employed to incorporate
vaccine and T cell costimulatory fusion proteins into a single
vector. Specifically, the original gp96-Ig vector was re-engineered
to generate a cell-based combination IO product that secretes both
the gp96-Ig fusion protein and various T cell costimulatory fusion
proteins (FIGS. 1 and 2). The combined local secretion of vaccine
and costimulatory fusion protein (FIG. 3) can activate tumor
antigen specific T cells, and is anticipated to enhance
antigen-specific immunity with limited cost and systemic toxicity,
particularly when combined with administration of an agent (e.g.,
an antibody against PD-1) that inhibits immunosuppressive molecules
produced by tumor cells.
Example 3
In vivo Studies of ImPACT vs. ComPACT
Materials and Methods
[0150] Cell culture and vaccine cell line generation: 3T3 cells
were maintained in IMDM with glutamine and 10% Bovine Growth Serum
(BGS) at 37.degree. C. in 5% CO.sub.2. A 3T3-Ovalbumin-Hygro
parental cell line was established using hygromycin resistant
plasmid backbone pcDNA3.1 encoding chicken ovalbumin (Ova) through
nucleofection with the 4D-NUCLEOFECTOR.TM. and Cell Line
NUCLEOFECTOR.TM. Kit SE (Lonza) according to the manufacturer's
directions. Single cell clones secreting high-level Ova were
screened by ELISA and used to generate 3T3-Ova-Gp96-Ig (ImPACT) and
3T3-Ova-Gp96-Ig/OX40L-Fc (ComPACT) through nucleofection of G418
resistant plasmid pB45 encoding either murine Gp96-Ig or Gp96-Ig
and the extracellular domain of OX40L-Fc, respectively. Again,
single cell clones of both ImPACT and ComPACT were generated
through antibiotic selection and clones secreting similar levels of
mouse IgG were screened further and used for subsequent analysis.
OX40L mRNA expression was confirmed by qRT-PCR and protein levels
were assessed by western blot.
[0151] CT26 cells were maintained in IMDM with glutamine and 10%
Fetal Bovine Serum at 37.degree. C. in 5% CO.sub.2. CT26 versions
of ImPACT (CT26-Gp96-Ig) and ComPACT (CT26-Gp96-Ig/OX40L-Fc) were
generated using the same expression plasmids as above, however
transfected into the CT26 cell line using EFFECTENE.RTM.
Transfection Reagents (Qiagen) according to manufacturer's
directions. Single cell clones were isolated under antibiotic
selection and screened for mouse IgG secretion by ELISA. OX40L mRNA
expression was confirmed by qRT-PCR.
[0152] B16.F10 cell lines were first established by generating an
ova parental clone (B16.F10-ova: as described above for 3T3 cells).
Then, B16.F10-ova versions of ImPACT (B16.F10-ova-gp96-Ig) and
ComPACT (B16.F10-ova-gp96-Ig/Fc-OX40L) were again transfected with
the identical plasmids as described above, and selected for
high-level gp96-Ig secretion.
[0153] Mouse models, OT-I/OT-II transfer and analysis: Antigen
specific CD8 T cells were isolated from the spleens of OT-I/EGFP
mice, carrying the T cell receptor transgenes TCR.alpha.-V2 and
TCR.beta.-V5, that recognize ovalbumin residues 257-264 during
H2K.sup.b MHC class I antigen cross-presentation. Antigen specific
CD4 T cells were isolated from the spleens of OT-II mice,
expressing the mouse .alpha.- and .beta.-chain T cell receptor that
is complementary with the CD4 co-receptor and is specific for
chicken ovalbumin residues 323-339 during I-A.sup.b MHC class II
antigen cross-presentation.
[0154] Briefly, mice were sacrificed through CO.sub.2 asphyxiation
followed by cervical dislocation, and the spleen was dissected into
sterile PBS+2 mM EDTA. Splenocytes were dissociated from the tissue
and passed through a 100 .mu.M strainer. Cells were pelleted at
1,200 RPM for 5 minutes and red blood cells were lysed by adding 5
mL 1.times.ACK lysis buffer (150 mM NH.sub.4Cl, 10 mM KHCO.sub.3
and 1 mM EDTA) for 1-2 minutes at room temperature. Following
lysis, an equal volume of 1.times.PBS was added and the cells were
again pelleted at 1,200 RPM for 5 minutes. OT-I (CD8) and OT-II
(CD4) were isolated from total splenocytes using CD4 and CD8
isolation kits from StemCell Technologies, according to the
manufacturer's directions. OT-I (0.5.times.10.sup.6 cells per
mouse) and OT-II (1.times.10.sup.6 cells per mouse) were
transferred via intravenous (IV) tail vein injections to mice
transgenic for FOXP3-RFP (to track regulatory T cells: Tregs). The
IV injection day corresponded to experimental day -1.
[0155] On days 0 and 35 (in the case of boosted mice), mice were
either untreated, vaccinated with the 3T3-Ova parental clone as a
control, vaccinated with ImPACT (alone or in combination with 100
.mu.g of agonist antibodies to ICOS (BioLegend #313512), 4-1BB (3H3
antibody, Bio-X-Cell) or OX40 (OX86 antibody, Bio-X-Cell), or
vaccinated with ComPACT. Vaccinations consisted of 1.times.10.sup.6
cells and were administered by intraperitoneal injection (IP).
Lymphocytes harvested from peripheral blood were analyzed by flow
cytometry throughout the time-course.
[0156] CT26 Tumor Model and Analysis: For CT26 tumor studies,
BALB/C mice were inoculated with either 2.times.10.sup.5 or
5.times.10.sup.5 tumor cells via subcutaneous injection into the
rear flank, indicating day 0. For B16.F10-ova studies, C57BL/6 mice
were inoculated with 5.times.10.sup.5 tumor cells into the rear
flank, indicating day 0. On vaccination days, tumor bearing mice
were either untreated or vaccinated with mitomycin-C (Sigma)
treated ImPACT, ImPACT+100 mg anti-OX86 (referred to as OX40(ab)
throughout) or ComPACT cells. Tumor area (mm.sup.2) and overall
survival was assessed throughout the time course. 30-day survival
criteria included total tumor area less than 175 mm.sup.2 with no
sign of tumor ulceration. Complete responders, in which tumors
established and were subsequently rejected following treatment, are
listed in FIG. 12, panel D and FIG. 11, panel E. A cohort of mice
inoculated with 2.times.10.sup.5 cells was sacrificed 11 days after
tumor inoculation. Tumors were excised from these mice, trypsinized
at 37.degree. C. for 10 minutes, dissociated, and passed through a
100 .mu.M cell strainer. Cells were pelleted, red blood cells were
lysed (as described above), and RNA was isolated, reverse
transcribed, and analyzed by qPCR (see below). A cohort of CT26
experimental mice was euthanized on day 12 for AH1-tetramer
analysis in splenocytes and genetic analysis of tumor tissue.
Tumors were excised from these mice, trypsinized at 37.degree. C.
for 10-15 minutes, dissociated and homogenized through a 100 mM
strainer. Cells were pelleted and processed for RNA isolation (see
below).
[0157] Flow Cytometry: Flow cytometry and cell sorting was
performed on the Sony SH800. For extracellular staining, cell
pellets were resuspended in 1.times.PBS buffer containing 1% bovine
serum albumin (BSA), 0.02% sodium azide, and 2 mM EDTA, and the
appropriate antibodies and incubated on ice in the dark for 30
minutes. Cells were then washed in flow cytometry buffer,
resuspended and then analyzed. For intracellular staining, cells
were fixed and permeabilized using the FOXP3 Fix/Perm kit from
BioLegend, stained as described above, washed in flow cytometry
buffer, resuspended and then analyzed. Antibodies used were
PE/Cy7-CD4 (Sony, 1102640), AF700-CD8a (Sony, 1103650), APC-TCR
V.beta.5.1,5.2 (Sony, 1297530), PacificBlue-TCR V.alpha.2 (Sony,
1239080), APC-KLRG1 (BioLegend, 138412), BV421-CD44 (BioLegend,
103039), BV605-CD127 (BioLegend, 135025), APC-Ki67 (BioLegend,
652406), PE/Cy7-IFN.gamma. (Biolegend, 505826), and BV421-IL2
(BioLegend, 503825).
[0158] ELISAs: Standard ELISA conditions were set such that
1.times.10.sup.6 cells were plated in 1 mL of culture media and the
supernatant analyzed after 24 hours. High-binding ELISA plates were
coated with 10 .mu.g/mL mouse IgG (Jackson Laboratories
#115-005-062) in sodium bicarbonate buffer. Coated plates were
incubated over night at 4.degree. C. The following morning, plates
were washed 3 times with TBS-T (50 mM Tris, 150 mM NaCl and 0.05%
Tween 20), blocked for 1 hour with Casein Blocking Buffer (Sigma)
and again washed 3 times with TBS-T. To the plates, 50 .mu.L of
cell supernatants along with an 11-point mouse IgG standard set of
samples were added to the coated ELISA plates and incubated at room
temperature for 1 hour. Plates were washed 3 times with TBS-T and
50 .mu.L of detection antibody (Jackson Laboratories #115-035-071)
was added, and incubated for 1 hour at room temperature in the
dark. Plates were washed 3 times with TBS-T and 100 .mu.L of
SUREBLUE.TM. TMB Microwell Peroxidase Substrate (KPL) was added to
each well and allowed to incubate at room temperature for 20
minutes in the dark. To stop the reaction, 100 .mu.L of sulfuric
acid was added to each well and plates were read immediately on a
BioTek plate reader. Samples were run at least in triplicate at
multiple dilutions.
[0159] RNA isolation and qRT-PCR: Total RNA was prepared using
RNeasy and RNeasy Micro kits (Qiagen) according to the
manufacturer's recommendations, including on-column DNase
treatment. A total of 1 .mu.g (using RNeasy) or 100 ng (using
RNeasy Micro) was used to synthesize cDNA with the First-strand
cDNA synthesis kit from OriGene. qPCR was performed using KAPA SYBR
FAST, SYBR green master mix (Kapa Biosystems) and then analyzed on
a Roche Lightcycler. Values were normalized to 18S mRNA and
represent the average.+-.standard error of the mean (SEM) for a
minimum of 3 biological replicates, all run in triplicate. Primer
sequences used were:
TABLE-US-00013 IFN-gamma: (SEQ ID NO: 14) F:
5'-CTGCCACGGCACAGTCATTG-3' (SEQ ID NO: 15) R:
5'-gccagttcctccagatatcc-3' TNF-alpha: (SEQ ID NO: 16) F:
5'-CCACGCTCTTCTGTCTACTG-3' (SEQ ID NO: 17) R:
3'-gccatagaactgatgagaggg-3' granzyme-B (SEQ ID NO: 18) F:
5'-CTACTGCTGACCTTGTCTCTG-3' (SEQ ID NO: 19) R:
3'-agtaaggccatgtagggtcg-3' IL-2 (SEQ ID NO: 20) F:
5'-CTGCGGCATGTTCTGGATTTGACT-3' (SEQ ID NO: 21) R:
5'-AGTCCACCACAGTTGCTGACTCAT-3' perforin-1 (SEQ ID NO: 22) F:
5'-GACACAGTAGAGTGTCGCATG-3' (SEQ ID NO: 23) R:
5'-aagcatgctctgtggagctg-3' beta-actin (SEQ ID NO: 24) F:
5'-aaggccaaccgtgaaaagat-3' (SEQ ID NO: 25) R:
5'-gtggtacgaccagaggcatac3'
[0160] Western Blot Analysis: ImPACT and ComPACT cells were treated
for 16 hours with Brefeldin-A to inhibit protein transport and
secretion. Cells were then lysed in RIPA buffer (25 mM Tris-HCL,
150 mM NaCl, 1% NP-40, 1% NaDeoxycholate, and 0.1% SDS), containing
1.times.complete protease inhibitor cocktail (Roche) for 10 minutes
on ice. Protein concentration was determined using DC Protein Assay
kit (Bio-Rad) and 20 .mu.g of protein was probed. Antibodies were:
CD252 (OX40L, Abcam #ab156285, 1:1000 dilution), histone H3 (Active
Motif #61278, 1:10,000), histone H4 (Active Motif #61300,
1:10,000), and beta actin (Abcam #ab8226, 1:10,000).
[0161] LEGENDplex Cytokine Analysis: Experimental mice were
euthanized through CO.sub.2 asphyxiation and cervical dislocation
and whole blood was collected via cardiac puncture. Red blood cells
were allowed to settle by gravity for 1 hour at room temperature
and the remaining cells were pelleted at 1,200 RPM for 5 minutes.
Serum was then transferred to a new 1.5 mL eppendorf tube. Cytokine
analysis was performed using the LEGENDPLEX.TM. Cytokine Analysis
kit (BioLegend) according to manufacturer recommendations and
analyzed on the Sony SH800.
[0162] Statistical Analysis: Experimental replicates (N) are shown
in the figures. Unless noted otherwise, values plotted represent
the mean from a minimum of 3 distinct experiments and error is SEM.
Statistical significance (p-value) was determined using unpaired
parametric t-tests with Welch's correction. Significant p-values
are labeled with an asterisk (*), and the corresponding p-value is
labeled in each figure.
Results
[0163] Many new trials will investigate whether adding a
therapeutic vaccine or T cell costimulatory antibody is an
effective strategy to increase the proportion of responding
patients and the durability of clinical responses. The
implementation of such a strategy is limited by several factors,
including an incomplete understanding of which agents may provide
synergistic benefit, whether to toxicities of such combinations
will be tolerable and eventually how the healthcare system will
manage such combinations.
[0164] To investigate the potential synergy between a vaccine and
individual T cell costimulatory molecules, a series of head-to-head
studies was performed in pre-clinical mouse models. Using a
cell-based vaccine expressing a modified secretable gp96-Ig fusion
protein (FIG. 4A), studies were conducted to investigate whether
co-administration of agonistic antibodies targeting OX40, 4-1BB, or
ICOS would provide further costimulation of antigen-specific CD8+ T
cells (FIGS. 5A-5C) Immunization of C57BL/6 mice that were
adoptively transferred with ovalbumin-specific CD8+ T cells (OT-I)
with a 3T3-ova-gp96-Ig vaccine led to proliferation of OT-I cells
to 10% of peripheral blood CD8+ T cells. This response could be
doubled by additional administration of OX40 agonist antibodies,
but not 4-1BB or ICOS co-stimulatory antibodies (FIG. 5D).
[0165] T cell costimulation by OX40L is triggered by local
inflammation in a spatially restricted microenvironment by antigen
presenting cells over the course of 2-5 days. Administration of
OX40 receptor agonist antibodies provides systemic costimulation
that can persist for several weeks. Since vaccines are typically
administered locally, experiments were performed to determine
whether an OX40L fusion protein (Fc-OX40L) could be co-expressed in
the second cassette of the gp96-Ig containing plasmid as a strategy
to both limit systemic co-stimulation and enable combination
immunotherapy with a single compound (FIG. 6A). As proof of
concept, a 3T3 cell co-expressing soluble ovalbumin and either
gp96-Ig alone ("ImPACT") or gp96-Ig together with Fc-OX40L, ICOSL,
or 4-1BBL was generated. These cell lines were stably selected to
secrete similar amounts of both ova and gp96-Ig (FIGS. 7A and 7B).
Expression of Fc-OX40L, ICOSL, or 4-1BBL was evaluated by RT-PCR
and Western blotting (FIGS. 7C and 7D), and shown to be
functionally active in cell culture supernatants by an IL-2
secretion assay from primary splenocytes.
[0166] The in vivo activity of ImPACT either alone or in
combination with OX40 agonist antibodies was compared to ComPACT
using the OT-I model described in FIG. 5. Distinct cell lines were
used in this experiment because the co-transfections described
above were not possible with the neomycin resistance cassette
expressing ova in FIG. 5. Since ComPACT was administered locally,
one might not have expected the dramatic priming and boosting
effects vs. the ImPACT combination with the OX40 agonist antibody,
which was administered systemically. As shown in FIGS. 6B and 6C,
however, ComPACT immunization provided surprisingly and
significantly improved proliferation of OT-I cells following
primary immunization either with ImPACT alone or in combination
with OX40 agonist antibodies. The peak expansion in the peripheral
blood was increased on day 5 with ComPACT, but more importantly, so
was the duration of the response from days 6-20.
[0167] The memory response to OX40 agonistic antibodies in
combination with vaccination is relatively weak within the antigen
specific CD8 compartment. The boost response was evaluated by
re-immunizing mice on day 35 after the primary immunization (FIG.
6C). While the combination of OX40 agonist antibodies provided a
relatively weak boost of the OT-I response, ComPACT treated mice
demonstrated a boost response that nearly matched the magnitude of
the primary response (FIG. 6C). Flow cytometric analysis of
splenocytes and peritoneal cells from mice receiving ComPACT,
revealed a marked increase in CD127.sup.+KLRG1.sup.- cells compared
to the other groups, indicating an increase in memory precursor
cells (FIG. 8B). This effect was observed with various ComPACTS,
including ComPACT (OX40L), ComPACT (ICOSL) and ComPACT (4-1BBL) but
not with OX40 agonist antibody treatment. The various ComPACTs did
not induce an increase in short-lived effector cells
(CD127.sup.-KLRG1.sup.+, FIG. 8B), as did OX40 agonist antibody
treatment. The ComPacts, however, did increase memory T cells
(CD127.sup.+KLRG1.sup.+) within the spleen (FIG. 8B). These data
indicate that local administration of an OX40L, ICOSL, or 4-1BBL
agonist fusion proteins significantly increased both the primary
and the boost response in the antigen-specific CD8 compartment,
which is correlated with an increase in memory precursor cells and
a prolonged contraction phase following priming In addition, these
data also revealed a novel and unexpected mechanism of action for
ComPACT treated mice in comparison to ImPACT +/-OX40 agonist
antibody.
[0168] It was possible that the reason for increased primary and
boost responses in the antigen-specific CD8 compartment with
locally provided OX40L was due to decreased off-target activation
provided by systemic administration of OX40 agonist antibodies. To
test this hypothesis, peritoneal cells, splenocytes and tumor
draining lymph node (TDLN) cells were isolated on day 8 from mice
that were immunized with ImPACT +/-OX40 agonist antibody or ComPACT
and analyzed by flow cytometry and quantitative RT-PCR (qRT-PCR) to
distinguish between off-target immune activation and an
antigen-specific response. Analysis of peritoneal cells isolated on
day 8 following primary immunization indicated increased numbers of
total mononuclear, OT-I, and OT-II cells in ComPACT treated mice,
but also increased numbers of total CD4 cells in mice treated with
OX40 agonist antibodies (FIG. 8A). Increased levels of total CD4+
cells and FOXP3+ regulatory T cells (Treg) were detected in the
peritoneal cavity, spleen and TDLN in mice treated with OX40
agonist antibodies (FIG. 8A and 8E). In contrast, ComPACT treated
mice specifically amplified antigen-specific OT-I (CD8+) and OT-II
(CD4+) cells with no apparent stimulation of Treg cells (FIGS. 8A
and 8E). Similar findings also were observed in the spleen and
lymph nodes, indicating systemic expansion of total CD4 cells as
well as antigen-specific CD4 cells (FIGS. 9A and 9B). CD4+FoxP3+
regulatory T cells (Treg) also were increased for OX40 agonist, but
not ComPACT treated animals Serum cytokine analysis further
demonstrated a systemic increase in IFN.gamma., TNF.alpha., IL-5
and IL-6 in mice treated with OX40 agonist antibodies (FIG. 8C). To
investigate the cellular source of the systemic cytokine increase,
RT-PCR was performed on either total CD8+ cells or OT-I cells on
day 8 following immunization. ComPACT treated mice showed an
increase in IFN.gamma., TNF.alpha. and granzyme-B that was isolated
to the OT-I population, whereas mice treated with OX40 agonist
antibodies showed an increase in both the OT-I and the total CD8
population (FIG. 8D).
[0169] These data indicate that OX40L fusion proteins can be
locally provided by stable transfection of a plasmid co-expressing
a heat shock protein gp96-Ig based vaccine. Initial feasibility
related to whether sufficient concentrations of Fc-OX40L were
secreted to provide costimulation demonstrated that this was
achievable, and surprisingly, more effective than systemic
administration of OX40 agonist antibodies. The costimulated OT-I
cells produced equivalent levels of effector cytokines as OX40
antibody costimulated OT-I cells, and would be expected to exert
increased cytotoxic activity against a target cell.
[0170] To investigate the functional activity of ImPACT +/-OX40
antibodies versus ComPACT in a murine tumor model, CT26 cells were
stably transfected with these constructs as outlined for 3T3 cells
in FIG. 7 (FIGS. 10A-10C). In one set of experiments, mice were
inoculated with CT26 cells on day 0, and then treated with
mitomycin-C treated CT26 cells, CT26-gp96-Ig, CT26-gp96-Ig combined
with OX40 agonist antibodies or with CT26 ComPACT on days 6 and 11
post tumor inoculation. In a second set of experiments, mice were
inoculated with CT26 cells on day 0, and then immunized with
mitomycin-C treated CT26 cells, CT26-ImPACT, CT26-ImPACT combined
with OX40 agonist antibody or with CT26-ComPACT cells on days 4, 7
and 10 post tumor inoculation (FIG. 11A). Quantitative RT-PCR on
tumor tissue isolated on day 12 post-tumor inoculation revealed
increased expression of CD8a, IL-2 and IFN.gamma. in OX40 agonist
antibody, ImPACT, ComPACT and ImPACT+OX40 agonist antibody
combination treated groups, indicating immune cell activation and
tumor infiltration. As expected, only mice receiving OX40 agonist
antibodies (either alone or together with ImPACT) showed increased
CD4 and FoxP3 expression within the tumor (FIG. 11B). CT26
antigen-specific CD8+ expansion, as detected by AH1-tetramer
staining, was significantly elevated approximately 4-fold in
ImPACT+OX40 antibody and approximately 5-fold in ComPACT treated
mice compared with the untreated group (FIG. 11B). Tumor
progression was shown to be strongly blocked in mice receiving
either ImPACT+OX40 agonist or ComPACT treatments as compared to the
control or monotherapy arms (FIG. 11D). This led to a significant
increase in long-term survival and a higher rate of complete tumor
rejection in ComPACT treated mice (FIG. 11E, 80% and approximately
47%, respectively) compared to what we observed with the B16.F10
tumor model. Accordingly, ComPACT generates potent antigen-specific
T cell expansion and tumor infiltration, delays in tumor growth and
significant survival benefits.
[0171] The B16.F10 mouse melanoma tumor model is an aggressive
tumor and is not typically treated effectively with OX40 agonist
antibody. In order to assess gp96-Ig based vaccines in the B16.F10
tumor model, a B16.F10-ova cell line was generated. In addition,
B16.F10-ova-ImPACT and--ComPACT vaccines were subsequently
generated by stable transfection of gp96-Ig and gp96-Ig-Fc-OX40L
vectors, respectively. Comparable levels of gp96-Ig secretion from
the B16.F10-ImPACT and--ComPACT cell lines were confirmed by ELISA
and Fc-OX40L expression in the B16.F10-ova-ComPACT cell line was
also confirmed by qRT-PCR. Mice were adoptively transferred with
OT-I cells a day prior to B16.F10-ova tumor inoculation (indicating
day -1, FIG. 12A). Next, the antigen specific response of OT-I
cells was investigated in mice following vaccination on days 4, 7
and 10 with mitomycin-C treated B16. F10-ova cells, B16.
F10-ova-ImPACT, B16. F10-ova-ImPACT combined with OX40 agonist
antibodies or with B16.F10-ova-ComPACT (FIG. 12B). Consistent with
the data obtained with the 3T3-ova model system,
B16.F10-ova-ComPACT treated mice exhibited a robust expansion of
OT-I cells between days 10 and 19 (which corresponded to days 6
through 15 following the initial vaccination), that was greater
than that seen with ImPACT +/-OX40 agonist antibodies, with similar
durable kinetics in the contraction phase to what was observed
previously. Accordingly, B16.F10-ova-ComPACT vaccinated mice
displayed a more potent anti-tumor effect than both ImPACT +/-OX40
agonist antibody vaccinations (FIG. 12C). The long term survival in
ComPACT treated mice was approximately 78%, with 11% of the mice
demonstrating complete rejection of their aggressive tumors. In
comparison ImPACT alone vaccinated mice and ImPACT+OX40 agonist Ab
treated mice showed overall survival rates of 50% and 62.5%,
respectively (FIG. 12D).
[0172] The functional activities of additional ComPACTs were
investigated using the previously described immunization assay as
well as the OT-I transfer assay. Specifically, OT-1/GFP cells were
analyzed by flow cytometry in mice treated with No Vaccine, Ova
only control cells, ComPACT (gp96-Ig/OX40L or gp96-Ig/TL1A) or
ComPACT.sup.2 (gp96-Ig/OX40L+TL1A), which is a mixture of a
ComPACT-OX40L cell line and a ComPACT-TL1A cell line, over 46 days,
with initial vaccination on day 0 and a boost on day 35 (FIG. 13).
Both prime and memory responses were strong in mice treated with
ComPACT (96-Ig/OX40L or gp96-Ig/TL1A) or ComPACT.sup.2
(gp96-Ig/OX40L+TL1A). ComPACT or ComPACT.sup.2 mice also
surprisingly retained elevated OT-1 levels throughout the
time-course (.about.days 7-20). Additionally, C57BL/6 mice were
immunized with ImPACT alone or ComPACT (gp96-OX40L, gp96-Ig/4-1BBL
or gp96-Ig/ICOS-L) at day 0 (FIG. 14). Results indicate that the
various ComPACTS enhanced the proliferation of OTI cells compared
to ImPACT.
[0173] The in vivo activities of the additional ComPACTS were
further investigated in the CT26 colorectal carcinoma model.
Specifically, mice were either untreated or vaccinated on days 4, 7
and 10 with CT26 parental cells, ImPACT alone, ImPACT+the TNFRSF25
agonist (4C12 ab), 4C12 (ab) alone, PD-1 (ab) alone, 4C12 (ab) and
PD-1 (ab), ComPACT (gp96-Ig/OX40L or gp96-Ig/TL1A), ComPACT
(gp96-Ig/OX40L) + PD-1 (ab), or ComPACT.sup.2 (gp96-Ig/OX40L+TL1A)
(FIG. 15). Results indicate that ComPACT treatment alone
(gp96-Ig/OX40L or gp96-Ig/TL1A) and in combination with PD-1
significantly reduced tumor growth. As shown in FIG. 16, ComPACT
treatment alone (gp96-Ig/OX40L or gp96-Ig/TL1A) and in combination
with PD-1 also significantly enhanced mice survival.
[0174] Expression of ComPACT in human cancer cell lines was tested.
Specifically, ComPACT (gp96-Ig/OX40L) was transfected into a human
prostate cancer cell line (e.g., PC-3) or a human lung
adenocarcinoma cell line (e.g., AD100). See FIGS. 17 and 18,
respectively. Results indicate that both cell lines produced and
excreted OX40L.
[0175] Altogether, these data demonstrated that combination
immunotherapy may be approached by incorporating multiple
complementary modalities, in this case a vaccine and T cell
costimulatory fusion protein, in a single compound. Provision of T
cell costimulation by vector-encoded and cell-secreted Fc-OX40L was
feasible, and led to enhanced proliferation of antigen-specific
CD8+ T cells at the time of both priming and boosting as compared
to OX40 agonistic antibodies. T cells activated by the combined
vaccine and costimulator produced IFN.gamma., IL-2, TNF.alpha., and
granzyme-B, and were not accompanied by off-target T cell
proliferation and systemic inflammatory cytokine increases observed
with OX40 agonist antibodies. Importantly, this approach also
enhanced therapeutic tumor immunity in an established murine colon
cancer model. Together, these results provide a strategy for
implementing combination immunotherapy that may not rely on double
or triple antibody combinations, and which may provide greater
safety and efficacy for patients due to reduced off-target T cell
activation.
Other Embodiments
[0176] It is to be understood that while the invention has been
described in conjunction with the detailed description thereof, the
foregoing description is intended to illustrate and not limit the
scope of the invention, which is defined by the scope of the
appended claims. Other aspects, advantages, and modifications are
within the scope of the following claims
[0177] The content of any individual section may be equally
applicable to all sections.
INCORPORATION BY REFERENCE
[0178] All patents and publications referenced herein are hereby
incorporated by reference in their entireties.
[0179] The publications discussed herein are provided solely for
their disclosure prior to the filing date of the present
application. Nothing herein is to be construed as an admission that
the present invention is not entitled to antedate such publication
by virtue of prior invention.
[0180] As used herein, all headings are simply for organization and
are not intended to limit the disclosure in any
Sequence CWU 1
1
4912412DNAHomo sapiens 1atgagggccc tgtgggtgct gggcctctgc tgcgtcctgc
tgaccttcgg gtcggtcaga 60gctgacgatg aagttgatgt ggatggtaca gtagaagagg
atctgggtaa aagtagagaa 120ggatcaagga cggatgatga agtagtacag
agagaggaag aagctattca gttggatgga 180ttaaatgcat cacaaataag
agaacttaga gagaagtcgg aaaagtttgc cttccaagcc 240gaagttaaca
gaatgatgaa acttatcatc aattcattgt ataaaaataa agagattttc
300ctgagagaac tgatttcaaa tgcttctgat gctttagata agataaggct
aatatcactg 360actgatgaaa atgctctttc tggaaatgag gaactaacag
tcaaaattaa gtgtgataag 420gagaagaacc tgctgcatgt cacagacacc
ggtgtaggaa tgaccagaga agagttggtt 480aaaaaccttg gtaccatagc
caaatctggg acaagcgagt ttttaaacaa aatgactgaa 540gcacaggaag
atggccagtc aacttctgaa ttgattggcc agtttggtgt cggtttctat
600tccgccttcc ttgtagcaga taaggttatt gtcacttcaa aacacaacaa
cgatacccag 660cacatctggg agtctgactc caatgaattt tctgtaattg
ctgacccaag aggaaacact 720ctaggacggg gaacgacaat tacccttgtc
ttaaaagaag aagcatctga ttaccttgaa 780ttggatacaa ttaaaaatct
cgtcaaaaaa tattcacagt tcataaactt tcctatttat 840gtatggagca
gcaagactga aactgttgag gagcccatgg aggaagaaga agcagccaaa
900gaagagaaag aagaatctga tgatgaagct gcagtagagg aagaagaaga
agaaaagaaa 960ccaaagacta aaaaagttga aaaaactgtc tgggactggg
aacttatgaa tgatatcaaa 1020ccaatatggc agagaccatc aaaagaagta
gaagaagatg aatacaaagc tttctacaaa 1080tcattttcaa aggaaagtga
tgaccccatg gcttatattc actttactgc tgaaggggaa 1140gttaccttca
aatcaatttt atttgtaccc acatctgctc cacgtggtct gtttgacgaa
1200tatggatcta aaaagagcga ttacattaag ctctatgtgc gccgtgtatt
catcacagac 1260gacttccatg atatgatgcc taaatacctc aattttgtca
agggtgtggt ggactcagat 1320gatctcccct tgaatgtttc ccgcgagact
cttcagcaac ataaactgct taaggtgatt 1380aggaagaagc ttgttcgtaa
aacgctggac atgatcaaga agattgctga tgataaatac 1440aatgatactt
tttggaaaga atttggtacc aacatcaagc ttggtgtgat tgaagaccac
1500tcgaatcgaa cacgtcttgc taaacttctt aggttccagt cttctcatca
tccaactgac 1560attactagcc tagaccagta tgtggaaaga atgaaggaaa
aacaagacaa aatctacttc 1620atggctgggt ccagcagaaa agaggctgaa
tcttctccat ttgttgagcg acttctgaaa 1680aagggctatg aagttattta
cctcacagaa cctgtggatg aatactgtat tcaggccctt 1740cccgaatttg
atgggaagag gttccagaat gttgccaagg aaggagtgaa gttcgatgaa
1800agtgagaaaa ctaaggagag tcgtgaagca gttgagaaag aatttgagcc
tctgctgaat 1860tggatgaaag ataaagccct taaggacaag attgaaaagg
ctgtggtgtc tcagcgcctg 1920acagaatctc cgtgtgcttt ggtggccagc
cagtacggat ggtctggcaa catggagaga 1980atcatgaaag cacaagcgta
ccaaacgggc aaggacatct ctacaaatta ctatgcgagt 2040cagaagaaaa
catttgaaat taatcccaga cacccgctga tcagagacat gcttcgacga
2100attaaggaag atgaagatga taaaacagtt ttggatcttg ctgtggtttt
gtttgaaaca 2160gcaacgcttc ggtcagggta tcttttacca gacactaaag
catatggaga tagaatagaa 2220agaatgcttc gcctcagttt gaacattgac
cctgatgcaa aggtggaaga agagcccgaa 2280gaagaacctg aagagacagc
agaagacaca acagaagaca cagagcaaga cgaagatgaa 2340gaaatggatg
tgggaacaga tgaagaagaa gaaacagcaa aggaatctac agctgaaaaa
2400gatgaattgt aa 24122803PRTHomo sapiens 2Met Arg Ala Leu Trp Val
Leu Gly Leu Cys Cys Val Leu Leu Thr Phe 1 5 10 15 Gly Ser Val Arg
Ala Asp Asp Glu Val Asp Val Asp Gly Thr Val Glu 20 25 30 Glu Asp
Leu Gly Lys Ser Arg Glu Gly Ser Arg Thr Asp Asp Glu Val 35 40 45
Val Gln Arg Glu Glu Glu Ala Ile Gln Leu Asp Gly Leu Asn Ala Ser 50
55 60 Gln Ile Arg Glu Leu Arg Glu Lys Ser Glu Lys Phe Ala Phe Gln
Ala 65 70 75 80 Glu Val Asn Arg Met Met Lys Leu Ile Ile Asn Ser Leu
Tyr Lys Asn 85 90 95 Lys Glu Ile Phe Leu Arg Glu Leu Ile Ser Asn
Ala Ser Asp Ala Leu 100 105 110 Asp Lys Ile Arg Leu Ile Ser Leu Thr
Asp Glu Asn Ala Leu Ser Gly 115 120 125 Asn Glu Glu Leu Thr Val Lys
Ile Lys Cys Asp Lys Glu Lys Asn Leu 130 135 140 Leu His Val Thr Asp
Thr Gly Val Gly Met Thr Arg Glu Glu Leu Val 145 150 155 160 Lys Asn
Leu Gly Thr Ile Ala Lys Ser Gly Thr Ser Glu Phe Leu Asn 165 170 175
Lys Met Thr Glu Ala Gln Glu Asp Gly Gln Ser Thr Ser Glu Leu Ile 180
185 190 Gly Gln Phe Gly Val Gly Phe Tyr Ser Ala Phe Leu Val Ala Asp
Lys 195 200 205 Val Ile Val Thr Ser Lys His Asn Asn Asp Thr Gln His
Ile Trp Glu 210 215 220 Ser Asp Ser Asn Glu Phe Ser Val Ile Ala Asp
Pro Arg Gly Asn Thr 225 230 235 240 Leu Gly Arg Gly Thr Thr Ile Thr
Leu Val Leu Lys Glu Glu Ala Ser 245 250 255 Asp Tyr Leu Glu Leu Asp
Thr Ile Lys Asn Leu Val Lys Lys Tyr Ser 260 265 270 Gln Phe Ile Asn
Phe Pro Ile Tyr Val Trp Ser Ser Lys Thr Glu Thr 275 280 285 Val Glu
Glu Pro Met Glu Glu Glu Glu Ala Ala Lys Glu Glu Lys Glu 290 295 300
Glu Ser Asp Asp Glu Ala Ala Val Glu Glu Glu Glu Glu Glu Lys Lys 305
310 315 320 Pro Lys Thr Lys Lys Val Glu Lys Thr Val Trp Asp Trp Glu
Leu Met 325 330 335 Asn Asp Ile Lys Pro Ile Trp Gln Arg Pro Ser Lys
Glu Val Glu Glu 340 345 350 Asp Glu Tyr Lys Ala Phe Tyr Lys Ser Phe
Ser Lys Glu Ser Asp Asp 355 360 365 Pro Met Ala Tyr Ile His Phe Thr
Ala Glu Gly Glu Val Thr Phe Lys 370 375 380 Ser Ile Leu Phe Val Pro
Thr Ser Ala Pro Arg Gly Leu Phe Asp Glu 385 390 395 400 Tyr Gly Ser
Lys Lys Ser Asp Tyr Ile Lys Leu Tyr Val Arg Arg Val 405 410 415 Phe
Ile Thr Asp Asp Phe His Asp Met Met Pro Lys Tyr Leu Asn Phe 420 425
430 Val Lys Gly Val Val Asp Ser Asp Asp Leu Pro Leu Asn Val Ser Arg
435 440 445 Glu Thr Leu Gln Gln His Lys Leu Leu Lys Val Ile Arg Lys
Lys Leu 450 455 460 Val Arg Lys Thr Leu Asp Met Ile Lys Lys Ile Ala
Asp Asp Lys Tyr 465 470 475 480 Asn Asp Thr Phe Trp Lys Glu Phe Gly
Thr Asn Ile Lys Leu Gly Val 485 490 495 Ile Glu Asp His Ser Asn Arg
Thr Arg Leu Ala Lys Leu Leu Arg Phe 500 505 510 Gln Ser Ser His His
Pro Thr Asp Ile Thr Ser Leu Asp Gln Tyr Val 515 520 525 Glu Arg Met
Lys Glu Lys Gln Asp Lys Ile Tyr Phe Met Ala Gly Ser 530 535 540 Ser
Arg Lys Glu Ala Glu Ser Ser Pro Phe Val Glu Arg Leu Leu Lys 545 550
555 560 Lys Gly Tyr Glu Val Ile Tyr Leu Thr Glu Pro Val Asp Glu Tyr
Cys 565 570 575 Ile Gln Ala Leu Pro Glu Phe Asp Gly Lys Arg Phe Gln
Asn Val Ala 580 585 590 Lys Glu Gly Val Lys Phe Asp Glu Ser Glu Lys
Thr Lys Glu Ser Arg 595 600 605 Glu Ala Val Glu Lys Glu Phe Glu Pro
Leu Leu Asn Trp Met Lys Asp 610 615 620 Lys Ala Leu Lys Asp Lys Ile
Glu Lys Ala Val Val Ser Gln Arg Leu 625 630 635 640 Thr Glu Ser Pro
Cys Ala Leu Val Ala Ser Gln Tyr Gly Trp Ser Gly 645 650 655 Asn Met
Glu Arg Ile Met Lys Ala Gln Ala Tyr Gln Thr Gly Lys Asp 660 665 670
Ile Ser Thr Asn Tyr Tyr Ala Ser Gln Lys Lys Thr Phe Glu Ile Asn 675
680 685 Pro Arg His Pro Leu Ile Arg Asp Met Leu Arg Arg Ile Lys Glu
Asp 690 695 700 Glu Asp Asp Lys Thr Val Leu Asp Leu Ala Val Val Leu
Phe Glu Thr 705 710 715 720 Ala Thr Leu Arg Ser Gly Tyr Leu Leu Pro
Asp Thr Lys Ala Tyr Gly 725 730 735 Asp Arg Ile Glu Arg Met Leu Arg
Leu Ser Leu Asn Ile Asp Pro Asp 740 745 750 Ala Lys Val Glu Glu Glu
Pro Glu Glu Glu Pro Glu Glu Thr Ala Glu 755 760 765 Asp Thr Thr Glu
Asp Thr Glu Gln Asp Glu Asp Glu Glu Met Asp Val 770 775 780 Gly Thr
Asp Glu Glu Glu Glu Thr Ala Lys Glu Ser Thr Ala Glu Lys 785 790 795
800 Asp Glu Leu 34PRTHomo sapiens 3Lys Asp Glu Leu 1
41455DNAArtificial SequenceSynthetic sequence 4atgagactgg
gaagccctgg cctgctgttt ctgctgttca gcagcctgag agccgacacc 60caggaaaaag
aagtgcgggc catggtggga agcgacgtgg aactgagctg cgcctgtcct
120gagggcagca gattcgacct gaacgacgtg tacgtgtact ggcagaccag
cgagagcaag 180accgtcgtga cctaccacat cccccagaac agctccctgg
aaaacgtgga cagccggtac 240agaaaccggg ccctgatgtc tcctgccggc
atgctgagag gcgacttcag cctgcggctg 300ttcaacgtga ccccccagga
cgagcagaaa ttccactgcc tggtgctgag ccagagcctg 360ggcttccagg
aagtgctgag cgtggaagtg accctgcacg tggccgccaa tttcagcgtg
420ccagtggtgt ctgcccccca cagcccttct caggatgagc tgaccttcac
ctgtaccagc 480atcaacggct accccagacc caatgtgtac tggatcaaca
agaccgacaa cagcctgctg 540gaccaggccc tgcagaacga taccgtgttc
ctgaacatgc ggggcctgta cgacgtggtg 600tccgtgctga gaatcgccag
aacccccagc gtgaacatcg gctgctgcat cgagaacgtg 660ctgctgcagc
agaacctgac cgtgggcagc cagaccggca acgacatcgg cgagagagac
720aagatcaccg agaaccccgt gtccaccggc gagaagaatg ccgccacctc
taagtacggc 780cctccctgcc cttcttgccc agcccctgaa tttctgggcg
gaccctccgt gtttctgttc 840cccccaaagc ccaaggacac cctgatgatc
agccggaccc ccgaagtgac ctgcgtggtg 900gtggatgtgt cccaggaaga
tcccgaggtg cagttcaatt ggtacgtgga cggggtggaa 960gtgcacaacg
ccaagaccaa gcccagagag gaacagttca acagcaccta ccgggtggtg
1020tctgtgctga ccgtgctgca ccaggattgg ctgagcggca aagagtacaa
gtgcaaggtg 1080tccagcaagg gcctgcccag cagcatcgaa aagaccatca
gcaacgccac cggccagccc 1140agggaacccc aggtgtacac actgccccct
agccaggaag agatgaccaa gaaccaggtg 1200tccctgacct gtctcgtgaa
gggcttctac ccctccgata tcgccgtgga atgggagagc 1260aacggccagc
cagagaacaa ctacaagacc acccccccag tgctggacag cgacggctca
1320ttcttcctgt actcccggct gacagtggac aagagcagct ggcaggaagg
caacgtgttc 1380agctgcagcg tgatgcacga agccctgcac aaccactaca
cccagaagtc cctgtctctg 1440tccctgggca aatga 14555484PRTArtificial
SequenceSynthetic sequence 5Met Arg Leu Gly Ser Pro Gly Leu Leu Phe
Leu Leu Phe Ser Ser Leu 1 5 10 15 Arg Ala Asp Thr Gln Glu Lys Glu
Val Arg Ala Met Val Gly Ser Asp 20 25 30 Val Glu Leu Ser Cys Ala
Cys Pro Glu Gly Ser Arg Phe Asp Leu Asn 35 40 45 Asp Val Tyr Val
Tyr Trp Gln Thr Ser Glu Ser Lys Thr Val Val Thr 50 55 60 Tyr His
Ile Pro Gln Asn Ser Ser Leu Glu Asn Val Asp Ser Arg Tyr 65 70 75 80
Arg Asn Arg Ala Leu Met Ser Pro Ala Gly Met Leu Arg Gly Asp Phe 85
90 95 Ser Leu Arg Leu Phe Asn Val Thr Pro Gln Asp Glu Gln Lys Phe
His 100 105 110 Cys Leu Val Leu Ser Gln Ser Leu Gly Phe Gln Glu Val
Leu Ser Val 115 120 125 Glu Val Thr Leu His Val Ala Ala Asn Phe Ser
Val Pro Val Val Ser 130 135 140 Ala Pro His Ser Pro Ser Gln Asp Glu
Leu Thr Phe Thr Cys Thr Ser 145 150 155 160 Ile Asn Gly Tyr Pro Arg
Pro Asn Val Tyr Trp Ile Asn Lys Thr Asp 165 170 175 Asn Ser Leu Leu
Asp Gln Ala Leu Gln Asn Asp Thr Val Phe Leu Asn 180 185 190 Met Arg
Gly Leu Tyr Asp Val Val Ser Val Leu Arg Ile Ala Arg Thr 195 200 205
Pro Ser Val Asn Ile Gly Cys Cys Ile Glu Asn Val Leu Leu Gln Gln 210
215 220 Asn Leu Thr Val Gly Ser Gln Thr Gly Asn Asp Ile Gly Glu Arg
Asp 225 230 235 240 Lys Ile Thr Glu Asn Pro Val Ser Thr Gly Glu Lys
Asn Ala Ala Thr 245 250 255 Ser Lys Tyr Gly Pro Pro Cys Pro Ser Cys
Pro Ala Pro Glu Phe Leu 260 265 270 Gly Gly Pro Ser Val Phe Leu Phe
Pro Pro Lys Pro Lys Asp Thr Leu 275 280 285 Met Ile Ser Arg Thr Pro
Glu Val Thr Cys Val Val Val Asp Val Ser 290 295 300 Gln Glu Asp Pro
Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu 305 310 315 320 Val
His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr 325 330
335 Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Ser
340 345 350 Gly Lys Glu Tyr Lys Cys Lys Val Ser Ser Lys Gly Leu Pro
Ser Ser 355 360 365 Ile Glu Lys Thr Ile Ser Asn Ala Thr Gly Gln Pro
Arg Glu Pro Gln 370 375 380 Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu
Met Thr Lys Asn Gln Val 385 390 395 400 Ser Leu Thr Cys Leu Val Lys
Gly Phe Tyr Pro Ser Asp Ile Ala Val 405 410 415 Glu Trp Glu Ser Asn
Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro 420 425 430 Pro Val Leu
Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr 435 440 445 Val
Asp Lys Ser Ser Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val 450 455
460 Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu
465 470 475 480 Ser Leu Gly Lys 61305DNAArtificial
SequenceSynthetic sequence 6atgtctaagt acggccctcc ctgccctagc
tgccctgccc ctgaatttct gggcggaccc 60agcgtgttcc tgttcccccc aaagcccaag
gacaccctga tgatcagccg gacccccgaa 120gtgacctgcg tggtggtgga
tgtgtcccag gaagatcccg aggtgcagtt caattggtac 180gtggacggcg
tggaagtgca caacgccaag accaagccca gagaggaaca gttcaacagc
240acctaccggg tggtgtccgt gctgaccgtg ctgcaccagg attggctgag
cggcaaagag 300tacaagtgca aggtgtccag caagggcctg cccagcagca
tcgagaaaac catcagcaac 360gccaccggcc agcccaggga accccaggtg
tacacactgc cccctagcca ggaagagatg 420accaagaacc aggtgtccct
gacctgtctc gtgaagggct tctacccctc cgatatcgcc 480gtggaatggg
agagcaacgg ccagcctgag aacaactaca agaccacccc cccagtgctg
540gacagcgacg gctcattctt cctgtacagc agactgaccg tggacaagag
cagctggcag 600gaaggcaacg tgttcagctg cagcgtgatg cacgaggccc
tgcacaacca ctacacccag 660aagtccctgt ctctgagcct gggcaaggcc
tgtccatggg ctgtgtctgg cgctagagcc 720tctcctggat ctgccgccag
ccccagactg agagagggac ctgagctgag ccccgatgat 780cctgccggac
tgctggatct gagacagggc atgttcgccc agctggtggc ccagaacgtg
840ctgctgatcg atggccccct gagctggtac agcgatcctg gactggctgg
cgtgtcactg 900acaggcggcc tgagctacaa agaggacacc aaagaactgg
tggtggccaa ggccggcgtg 960tactacgtgt tctttcagct ggaactgcgg
agagtggtgg ccggcgaagg atccggctct 1020gtgtctctgg ctctgcatct
gcagcccctg agatctgctg ctggcgctgc tgctctggcc 1080ctgacagtgg
acctgcctcc tgcctctagc gaggccagaa acagcgcatt cgggtttcaa
1140ggcagactgc tgcacctgtc tgccggccag agactgggag tgcatctgca
cacagaggcc 1200agagccaggc acgcctggca gctgactcag ggcgctacag
tgctgggcct gttcagagtg 1260acccccgaga ttccagccgg cctgcctagc
cccagatccg aatga 13057434PRTArtificial SequenceSynthetic sequence
7Met Ser Lys Tyr Gly Pro Pro Cys Pro Ser Cys Pro Ala Pro Glu Phe 1
5 10 15 Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp
Thr 20 25 30 Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val
Val Asp Val 35 40 45 Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp
Tyr Val Asp Gly Val 50 55 60 Glu Val His Asn Ala Lys Thr Lys Pro
Arg Glu Glu Gln Phe Asn Ser 65 70 75 80 Thr Tyr Arg Val Val Ser Val
Leu Thr Val Leu His Gln Asp Trp Leu 85 90 95 Ser Gly Lys Glu Tyr
Lys Cys Lys Val Ser Ser Lys Gly Leu Pro Ser 100 105 110 Ser Ile Glu
Lys Thr Ile Ser Asn Ala Thr Gly Gln Pro Arg Glu Pro 115 120 125 Gln
Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln 130 135
140 Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala
145 150 155 160 Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr
Lys Thr Thr 165 170 175 Pro Pro Val
Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu 180 185 190 Thr
Val Asp Lys Ser Ser Trp Gln Glu Gly Asn Val Phe Ser Cys Ser 195 200
205 Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
210 215 220 Leu Ser Leu Gly Lys Ala Cys Pro Trp Ala Val Ser Gly Ala
Arg Ala 225 230 235 240 Ser Pro Gly Ser Ala Ala Ser Pro Arg Leu Arg
Glu Gly Pro Glu Leu 245 250 255 Ser Pro Asp Asp Pro Ala Gly Leu Leu
Asp Leu Arg Gln Gly Met Phe 260 265 270 Ala Gln Leu Val Ala Gln Asn
Val Leu Leu Ile Asp Gly Pro Leu Ser 275 280 285 Trp Tyr Ser Asp Pro
Gly Leu Ala Gly Val Ser Leu Thr Gly Gly Leu 290 295 300 Ser Tyr Lys
Glu Asp Thr Lys Glu Leu Val Val Ala Lys Ala Gly Val 305 310 315 320
Tyr Tyr Val Phe Phe Gln Leu Glu Leu Arg Arg Val Val Ala Gly Glu 325
330 335 Gly Ser Gly Ser Val Ser Leu Ala Leu His Leu Gln Pro Leu Arg
Ser 340 345 350 Ala Ala Gly Ala Ala Ala Leu Ala Leu Thr Val Asp Leu
Pro Pro Ala 355 360 365 Ser Ser Glu Ala Arg Asn Ser Ala Phe Gly Phe
Gln Gly Arg Leu Leu 370 375 380 His Leu Ser Ala Gly Gln Arg Leu Gly
Val His Leu His Thr Glu Ala 385 390 395 400 Arg Ala Arg His Ala Trp
Gln Leu Thr Gln Gly Ala Thr Val Leu Gly 405 410 415 Leu Phe Arg Val
Thr Pro Glu Ile Pro Ala Gly Leu Pro Ser Pro Arg 420 425 430 Ser Glu
81284DNAArtificial SequenceSynthetic sequence 8atgtctaagt
acggccctcc ctgccctagc tgccctgccc ctgaatttct gggcggaccc 60agcgtgttcc
tgttcccccc aaagcccaag gacaccctga tgatcagccg gacccccgaa
120gtgacctgcg tggtggtgga tgtgtcccag gaagatcccg aggtgcagtt
caattggtac 180gtggacggcg tggaagtgca caacgccaag accaagccca
gagaggaaca gttcaacagc 240acctaccggg tggtgtccgt gctgaccgtg
ctgcaccagg attggctgag cggcaaagag 300tacaagtgca aggtgtccag
caagggcctg cccagcagca tcgagaaaac catcagcaac 360gccaccggcc
agcccaggga accccaggtg tacacactgc cccctagcca ggaagagatg
420accaagaacc aggtgtccct gacctgtctc gtgaagggct tctacccctc
cgatatcgcc 480gtggaatggg agagcaacgg ccagcctgag aacaactaca
agaccacccc cccagtgctg 540gacagcgacg gctcattctt cctgtacagc
agactgaccg tggacaagag cagctggcag 600gaaggcaacg tgttcagctg
cagcgtgatg cacgaggccc tgcacaacca ctacacccag 660aagtccctgt
ctctgagcct gggcaagatc gagggccgga tggatagagc ccagggcgaa
720gcctgcgtgc agttccaggc tctgaagggc caggaattcg cccccagcca
ccagcaggtg 780tacgcccctc tgagagccga cggcgataag cctagagccc
acctgacagt cgtgcggcag 840acccctaccc agcacttcaa gaatcagttc
cccgccctgc actgggagca cgaactgggc 900ctggccttca ccaagaacag
aatgaactac accaacaagt ttctgctgat ccccgagagc 960ggcgactact
tcatctacag ccaagtgacc ttccggggca tgaccagcga gtgcagcgag
1020atcagacagg ccggcagacc taacaagccc gacagcatca ccgtcgtgat
caccaaagtg 1080accgacagct accccgagcc cacccagctg ctgatgggca
ccaagagcgt gtgcgaagtg 1140ggcagcaact ggttccagcc catctacctg
ggcgccatgt ttagtctgca agagggcgac 1200aagctgatgg tcaacgtgtc
cgacatcagc ctggtggatt acaccaaaga ggacaagacc 1260ttcttcggcg
cctttctgct ctga 12849427PRTArtificial SequenceSynthetic sequence
9Met Ser Lys Tyr Gly Pro Pro Cys Pro Ser Cys Pro Ala Pro Glu Phe 1
5 10 15 Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp
Thr 20 25 30 Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val
Val Asp Val 35 40 45 Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp
Tyr Val Asp Gly Val 50 55 60 Glu Val His Asn Ala Lys Thr Lys Pro
Arg Glu Glu Gln Phe Asn Ser 65 70 75 80 Thr Tyr Arg Val Val Ser Val
Leu Thr Val Leu His Gln Asp Trp Leu 85 90 95 Ser Gly Lys Glu Tyr
Lys Cys Lys Val Ser Ser Lys Gly Leu Pro Ser 100 105 110 Ser Ile Glu
Lys Thr Ile Ser Asn Ala Thr Gly Gln Pro Arg Glu Pro 115 120 125 Gln
Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln 130 135
140 Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala
145 150 155 160 Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr
Lys Thr Thr 165 170 175 Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe
Leu Tyr Ser Arg Leu 180 185 190 Thr Val Asp Lys Ser Ser Trp Gln Glu
Gly Asn Val Phe Ser Cys Ser 195 200 205 Val Met His Glu Ala Leu His
Asn His Tyr Thr Gln Lys Ser Leu Ser 210 215 220 Leu Ser Leu Gly Lys
Ile Glu Gly Arg Met Asp Arg Ala Gln Gly Glu 225 230 235 240 Ala Cys
Val Gln Phe Gln Ala Leu Lys Gly Gln Glu Phe Ala Pro Ser 245 250 255
His Gln Gln Val Tyr Ala Pro Leu Arg Ala Asp Gly Asp Lys Pro Arg 260
265 270 Ala His Leu Thr Val Val Arg Gln Thr Pro Thr Gln His Phe Lys
Asn 275 280 285 Gln Phe Pro Ala Leu His Trp Glu His Glu Leu Gly Leu
Ala Phe Thr 290 295 300 Lys Asn Arg Met Asn Tyr Thr Asn Lys Phe Leu
Leu Ile Pro Glu Ser 305 310 315 320 Gly Asp Tyr Phe Ile Tyr Ser Gln
Val Thr Phe Arg Gly Met Thr Ser 325 330 335 Glu Cys Ser Glu Ile Arg
Gln Ala Gly Arg Pro Asn Lys Pro Asp Ser 340 345 350 Ile Thr Val Val
Ile Thr Lys Val Thr Asp Ser Tyr Pro Glu Pro Thr 355 360 365 Gln Leu
Leu Met Gly Thr Lys Ser Val Cys Glu Val Gly Ser Asn Trp 370 375 380
Phe Gln Pro Ile Tyr Leu Gly Ala Met Phe Ser Leu Gln Glu Gly Asp 385
390 395 400 Lys Leu Met Val Asn Val Ser Asp Ile Ser Leu Val Asp Tyr
Thr Lys 405 410 415 Glu Asp Lys Thr Phe Phe Gly Ala Phe Leu Leu 420
425 101107DNAArtificial SequenceSynthetic sequence 10atgtctaagt
acggccctcc ctgccctagc tgccctgccc ctgaatttct gggcggaccc 60agcgtgttcc
tgttcccccc aaagcccaag gacaccctga tgatcagccg gacccccgaa
120gtgacctgcg tggtggtgga tgtgtcccag gaagatcccg aggtgcagtt
caattggtac 180gtggacggcg tggaagtgca caacgccaag accaagccca
gagaggaaca gttcaacagc 240acctaccggg tggtgtccgt gctgaccgtg
ctgcaccagg attggctgag cggcaaagag 300tacaagtgca aggtgtccag
caagggcctg cccagcagca tcgagaaaac catcagcaac 360gccaccggcc
agcccaggga accccaggtg tacacactgc cccctagcca ggaagagatg
420accaagaacc aggtgtccct gacctgtctc gtgaagggct tctacccctc
cgatatcgcc 480gtggaatggg agagcaacgg ccagcctgag aacaactaca
agaccacccc cccagtgctg 540gacagcgacg gctcattctt cctgtacagc
agactgaccg tggacaagag cagctggcag 600gaaggcaacg tgttcagctg
cagcgtgatg cacgaggccc tgcacaacca ctacacccag 660aagtccctgt
ctctgagcct gggcaagatc gagggccgga tggatcaggt gtcacacaga
720tacccccgga tccagagcat caaagtgcag tttaccgagt acaagaaaga
gaagggcttt 780atcctgacca gccagaaaga ggacgagatc atgaaggtgc
agaacaacag cgtgatcatc 840aactgcgacg ggttctacct gatcagcctg
aagggctact tcagtcagga agtgaacatc 900agcctgcact accagaagga
cgaggaaccc ctgttccagc tgaagaaagt gcggagcgtg 960aacagcctga
tggtggcctc tctgacctac aaggacaagg tgtacctgaa cgtgaccacc
1020gacaacacca gcctggacga cttccacgtg aacggcggcg agctgatcct
gattcaccag 1080aaccccggcg agttctgcgt gctctga 110711368PRTArtificial
SequenceSynthetic sequence 11Met Ser Lys Tyr Gly Pro Pro Cys Pro
Ser Cys Pro Ala Pro Glu Phe 1 5 10 15 Leu Gly Gly Pro Ser Val Phe
Leu Phe Pro Pro Lys Pro Lys Asp Thr 20 25 30 Leu Met Ile Ser Arg
Thr Pro Glu Val Thr Cys Val Val Val Asp Val 35 40 45 Ser Gln Glu
Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val 50 55 60 Glu
Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser 65 70
75 80 Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp
Leu 85 90 95 Ser Gly Lys Glu Tyr Lys Cys Lys Val Ser Ser Lys Gly
Leu Pro Ser 100 105 110 Ser Ile Glu Lys Thr Ile Ser Asn Ala Thr Gly
Gln Pro Arg Glu Pro 115 120 125 Gln Val Tyr Thr Leu Pro Pro Ser Gln
Glu Glu Met Thr Lys Asn Gln 130 135 140 Val Ser Leu Thr Cys Leu Val
Lys Gly Phe Tyr Pro Ser Asp Ile Ala 145 150 155 160 Val Glu Trp Glu
Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr 165 170 175 Pro Pro
Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu 180 185 190
Thr Val Asp Lys Ser Ser Trp Gln Glu Gly Asn Val Phe Ser Cys Ser 195
200 205 Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu
Ser 210 215 220 Leu Ser Leu Gly Lys Ile Glu Gly Arg Met Asp Gln Val
Ser His Arg 225 230 235 240 Tyr Pro Arg Ile Gln Ser Ile Lys Val Gln
Phe Thr Glu Tyr Lys Lys 245 250 255 Glu Lys Gly Phe Ile Leu Thr Ser
Gln Lys Glu Asp Glu Ile Met Lys 260 265 270 Val Gln Asn Asn Ser Val
Ile Ile Asn Cys Asp Gly Phe Tyr Leu Ile 275 280 285 Ser Leu Lys Gly
Tyr Phe Ser Gln Glu Val Asn Ile Ser Leu His Tyr 290 295 300 Gln Lys
Asp Glu Glu Pro Leu Phe Gln Leu Lys Lys Val Arg Ser Val 305 310 315
320 Asn Ser Leu Met Val Ala Ser Leu Thr Tyr Lys Asp Lys Val Tyr Leu
325 330 335 Asn Val Thr Thr Asp Asn Thr Ser Leu Asp Asp Phe His Val
Asn Gly 340 345 350 Gly Glu Leu Ile Leu Ile His Gln Asn Pro Gly Glu
Phe Cys Val Leu 355 360 365 121588DNAHomo sapiens 12tcccaagtag
ctgggactac aggagcccac caccaccccc ggctaatttt ttgtattttt 60agtagagacg
gggtttcacc gtgttagcca agatggtctt gatcacctga cctcgtgatc
120cacccgcctt ggcctcccaa agtgctggga ttacaggcat gagccaccgc
gcccggcctc 180cattcaagtc tttattgaat atctgctatg ttctacacac
tgttctaggt gctggggatg 240caacagggga caaaataggc aaaatccctg
tccttttggg gttgacattc tagtgactct 300tcatgtagtc tagaagaagc
tcagtgaata gtgtctgtgg ttgttaccag ggacacaatg 360acaggaacat
tcttgggtag agtgagaggc ctggggaggg aagggtctct aggatggagc
420agatgctggg cagtcttagg gagcccctcc tggcatgcac cccctcatcc
ctcaggccac 480ccccgtccct tgcaggagca ccctggggag ctgtccagag
cgctgtgccg ctgtctgtgg 540ctggaggcag agtaggtggt gtgctgggaa
tgcgagtggg agaactggga tggaccgagg 600ggaggcgggt gaggaggggg
gcaaccaccc aacacccacc agctgctttc agtgttctgg 660gtccaggtgc
tcctggctgg ccttgtggtc cccctcctgc ttggggccac cctgacctac
720acataccgcc actgctggcc tcacaagccc ctggttactg cagatgaagc
tgggatggag 780gctctgaccc caccaccggc cacccatctg tcacccttgg
acagcgccca cacccttcta 840gcacctcctg acagcagtga gaagatctgc
accgtccagt tggtgggtaa cagctggacc 900cctggctacc ccgagaccca
ggaggcgctc tgcccgcagg tgacatggtc ctgggaccag 960ttgcccagca
gagctcttgg ccccgctgct gcgcccacac tctcgccaga gtccccagcc
1020ggctcgccag ccatgatgct gcagccgggc ccgcagctct acgacgtgat
ggacgcggtc 1080ccagcgcggc gctggaagga gttcgtgcgc acgctggggc
tgcgcgaggc agagatcgaa 1140gccgtggagg tggagatcgg ccgcttccga
gaccagcagt acgagatgct caagcgctgg 1200cgccagcagc agcccgcggg
cctcggagcc gtttacgcgg ccctggagcg catggggctg 1260gacggctgcg
tggaagactt gcgcagccgc ctgcagcgcg gcccgtgaca cggcgcccac
1320ttgccaccta ggcgctctgg tggcccttgc agaagcccta agtacggtta
cttatgcgtg 1380tagacatttt atgtcactta ttaagccgct ggcacggccc
tgcgtagcag caccagccgg 1440ccccacccct gctcgcccct atcgctccag
ccaaggcgaa gaagcacgaa cgaatgtcga 1500gagggggtga agacatttct
caacttctcg gccggagttt ggctgagatc gcggtattaa 1560atctgtgaaa
gaaaacaaaa caaaacaa 158813426PRTHomo sapiens 13Met Glu Gln Arg Pro
Arg Gly Cys Ala Ala Val Ala Ala Ala Leu Leu 1 5 10 15 Leu Val Leu
Leu Gly Ala Arg Ala Gln Gly Gly Thr Arg Ser Pro Arg 20 25 30 Cys
Asp Cys Ala Gly Asp Phe His Lys Lys Ile Gly Leu Phe Cys Cys 35 40
45 Arg Gly Cys Pro Ala Gly His Tyr Leu Lys Ala Pro Cys Thr Glu Pro
50 55 60 Cys Gly Asn Ser Thr Cys Leu Val Cys Pro Gln Asp Thr Phe
Leu Ala 65 70 75 80 Trp Glu Asn His His Asn Ser Glu Cys Ala Arg Cys
Gln Ala Cys Asp 85 90 95 Glu Gln Ala Ser Gln Val Ala Leu Glu Asn
Cys Ser Ala Val Ala Asp 100 105 110 Thr Arg Cys Gly Cys Lys Pro Gly
Trp Phe Val Glu Cys Gln Val Ser 115 120 125 Gln Cys Val Ser Ser Ser
Pro Phe Tyr Cys Gln Pro Cys Leu Asp Cys 130 135 140 Gly Ala Leu His
Arg His Thr Arg Leu Leu Cys Ser Arg Arg Asp Thr 145 150 155 160 Asp
Cys Gly Thr Cys Leu Pro Gly Phe Tyr Glu His Gly Asp Gly Cys 165 170
175 Val Ser Cys Pro Thr Pro Pro Pro Ser Leu Ala Gly Ala Pro Trp Gly
180 185 190 Ala Val Gln Ser Ala Val Pro Leu Ser Val Ala Gly Gly Arg
Val Gly 195 200 205 Val Phe Trp Val Gln Val Leu Leu Ala Gly Leu Val
Val Pro Leu Leu 210 215 220 Leu Gly Ala Thr Leu Thr Tyr Thr Tyr Arg
His Cys Trp Pro His Lys 225 230 235 240 Pro Leu Val Thr Ala Asp Glu
Ala Gly Met Glu Ala Leu Thr Pro Pro 245 250 255 Pro Ala Thr His Leu
Ser Pro Leu Asp Ser Ala His Thr Leu Leu Ala 260 265 270 Pro Pro Asp
Ser Ser Glu Lys Ile Cys Thr Val Gln Leu Val Gly Asn 275 280 285 Ser
Trp Thr Pro Gly Tyr Pro Glu Thr Gln Glu Ala Leu Cys Pro Gln 290 295
300 Val Thr Trp Ser Trp Asp Gln Leu Pro Ser Arg Ala Leu Gly Pro Ala
305 310 315 320 Ala Ala Pro Thr Leu Ser Pro Glu Ser Pro Ala Gly Ser
Pro Ala Met 325 330 335 Met Leu Gln Pro Gly Pro Gln Leu Tyr Asp Val
Met Asp Ala Val Pro 340 345 350 Ala Arg Arg Trp Lys Glu Phe Val Arg
Thr Leu Gly Leu Arg Glu Ala 355 360 365 Glu Ile Glu Ala Val Glu Val
Glu Ile Gly Arg Phe Arg Asp Gln Gln 370 375 380 Tyr Glu Met Leu Lys
Arg Trp Arg Gln Gln Gln Pro Ala Gly Leu Gly 385 390 395 400 Ala Val
Tyr Ala Ala Leu Glu Arg Met Gly Leu Asp Gly Cys Val Glu 405 410 415
Asp Leu Arg Ser Arg Leu Gln Arg Gly Pro 420 425 1420DNAArtificial
sequenceSynthetic sequence 14ctgccacggc acagtcattg
201520DNAArtificial sequenceSynthetic sequence 15gccagttcct
ccagatatcc 201620DNAArtificial sequenceSynthetic sequence
16ccacgctctt ctgtctactg 201721DNAArtificial sequenceSynthetic
sequence 17gccatagaac tgatgagagg g 211821DNAArtificial
sequenceSynthetic sequence 18ctactgctga ccttgtctct g
211920DNAArtificial sequenceSynthetic sequence 19agtaaggcca
tgtagggtcg 202024DNAArtificial sequenceSynthetic sequence
20ctgcggcatg ttctggattt gact 242124DNAArtificial sequenceSynthetic
sequence 21agtccaccac agttgctgac tcat 242221DNAArtificial
sequenceSynthetic sequence 22gacacagtag agtgtcgcat g
212320DNAArtificial sequenceSynthetic sequence 23aagcatgctc
tgtggagctg 202420DNAArtificial sequenceSynthetic sequence
24aaggccaacc gtgaaaagat 202521DNAArtificial sequenceSynthetic
sequence
25gtggtacgac cagaggcata c 21265PRTArtificial SequenceSynthetic
sequence 26Gly Gly Gly Gly Ser 1 5 275PRTArtificial
sequenceSynthetic sequence 27Gly Gly Gly Gly Ser 1 5
288PRTArtificial sequenceSynthetic sequence 28Gly Gly Gly Gly Gly
Gly Gly Gly 1 5 296PRTArtificial sequenceSynthetic sequence 29Gly
Gly Gly Gly Gly Gly 1 5 305PRTArtificial sequenceSynthetic sequence
30Glu Ala Ala Ala Lys 1 5 317PRTArtificial sequenceSynthetic
sequence 31Ala Glu Ala Ala Ala Lys Ala 1 5 3212PRTArtificial
sequenceSynthetic sequence 32Ala Glu Ala Ala Ala Lys Glu Ala Ala
Ala Lys Ala 1 5 10 3346PRTArtificial sequenceSynthetic sequence
33Ala Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys 1
5 10 15 Glu Ala Ala Ala Lys Ala Leu Glu Ala Glu Ala Ala Ala Lys Glu
Ala 20 25 30 Ala Ala Lys Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys
Ala 35 40 45 345PRTArtificial sequenceSynthetic sequence 34Pro Ala
Pro Ala Pro 1 5 3518PRTArtificial sequenceSynthetic sequence 35Lys
Glu Ser Gly Ser Val Ser Ser Glu Gln Leu Ala Gln Phe Arg Ser 1 5 10
15 Leu Asp 3614PRTArtificial sequenceSynthetic sequence 36Glu Gly
Lys Ser Ser Gly Ser Gly Ser Glu Ser Lys Ser Thr 1 5 10
3712PRTArtificial sequenceSynthetic sequence 37Gly Ser Ala Gly Ser
Ala Ala Gly Ser Gly Glu Phe 1 5 10 38852DNAHomo sapiens
38atggagcctc ctggagactg ggggcctcct ccctggagat ccacccccaa aaccgacgtc
60ttgaggctgg tgctgtatct caccttcctg ggagccccct gctacgcccc agctctgccg
120tcctgcaagg aggacgagta cccagtgggc tccgagtgct gccccaagtg
cagtccaggt 180tatcgtgtga aggaggcctg cggggagctg acgggcacag
tgtgtgaacc ctgccctcca 240ggcacctaca ttgcccacct caatggccta
agcaagtgtc tgcagtgcca aatgtgtgac 300ccagccatgg gcctgcgcgc
gagccggaac tgctccagga cagagaacgc cgtgtgtggc 360tgcagcccag
gccacttctg catcgtccag gacggggacc actgcgccgc gtgccgcgct
420tacgccacct ccagcccggg ccagagggtg cagaagggag gcaccgagag
tcaggacacc 480ctgtgtcaga actgcccccc ggggaccttc tctcccaatg
ggaccctgga ggaatgtcag 540caccagacca agtgcagctg gctggtgacg
aaggccggag ctgggaccag cagctcccac 600tgggtatggt ggtttctctc
agggagcctc gtcatcgtca ttgtttgctc cacagttggc 660ctaatcatat
gtgtgaaaag aagaaagcca aggggtgatg tagtcaaggt gatcgtctcc
720gtccagcgga aaagacagga ggcagaaggt gaggccacag tcattgaggc
cctgcaggcc 780cctccggacg tcaccacggt ggccgtggag gagacaatac
cctcattcac ggggaggagc 840ccaaaccatt aa 85239283PRTHomo sapiens
39Met Glu Pro Pro Gly Asp Trp Gly Pro Pro Pro Trp Arg Ser Thr Pro 1
5 10 15 Lys Thr Asp Val Leu Arg Leu Val Leu Tyr Leu Thr Phe Leu Gly
Ala 20 25 30 Pro Cys Tyr Ala Pro Ala Leu Pro Ser Cys Lys Glu Asp
Glu Tyr Pro 35 40 45 Val Gly Ser Glu Cys Cys Pro Lys Cys Ser Pro
Gly Tyr Arg Val Lys 50 55 60 Glu Ala Cys Gly Glu Leu Thr Gly Thr
Val Cys Glu Pro Cys Pro Pro 65 70 75 80 Gly Thr Tyr Ile Ala His Leu
Asn Gly Leu Ser Lys Cys Leu Gln Cys 85 90 95 Gln Met Cys Asp Pro
Ala Met Gly Leu Arg Ala Ser Arg Asn Cys Ser 100 105 110 Arg Thr Glu
Asn Ala Val Cys Gly Cys Ser Pro Gly His Phe Cys Ile 115 120 125 Val
Gln Asp Gly Asp His Cys Ala Ala Cys Arg Ala Tyr Ala Thr Ser 130 135
140 Ser Pro Gly Gln Arg Val Gln Lys Gly Gly Thr Glu Ser Gln Asp Thr
145 150 155 160 Leu Cys Gln Asn Cys Pro Pro Gly Thr Phe Ser Pro Asn
Gly Thr Leu 165 170 175 Glu Glu Cys Gln His Gln Thr Lys Cys Ser Trp
Leu Val Thr Lys Ala 180 185 190 Gly Ala Gly Thr Ser Ser Ser His Trp
Val Trp Trp Phe Leu Ser Gly 195 200 205 Ser Leu Val Ile Val Ile Val
Cys Ser Thr Val Gly Leu Ile Ile Cys 210 215 220 Val Lys Arg Arg Lys
Pro Arg Gly Asp Val Val Lys Val Ile Val Ser 225 230 235 240 Val Gln
Arg Lys Arg Gln Glu Ala Glu Gly Glu Ala Thr Val Ile Glu 245 250 255
Ala Leu Gln Ala Pro Pro Asp Val Thr Thr Val Ala Val Glu Glu Thr 260
265 270 Ile Pro Ser Phe Thr Gly Arg Ser Pro Asn His 275 280
404900DNAHomo sapiens 40taaagtcatc aaaacaacgt tatatcctgt gtgaaatgct
gcagtcagga tgccttgtgg 60tttgagtgcc ttgatcatgt gccctaaggg gatggtggcg
gtggtggtgg ccgtggatga 120cggagactct caggccttgg caggtgcgtc
tttcagttcc cctcacactt cgggttcctc 180ggggaggagg ggctggaacc
ctagcccatc gtcaggacaa agatgctcag gctgctcttg 240gctctcaact
tattcccttc aattcaagta acaggaaaca agattttggt gaagcagtcg
300cccatgcttg tagcgtacga caatgcggtc aaccttagct gcaagtattc
ctacaatctc 360ttctcaaggg agttccgggc atcccttcac aaaggactgg
atagtgctgt ggaagtctgt 420gttgtatatg ggaattactc ccagcagctt
caggtttact caaaaacggg gttcaactgt 480gatgggaaat tgggcaatga
atcagtgaca ttctacctcc agaatttgta tgttaaccaa 540acagatattt
acttctgcaa aattgaagtt atgtatcctc ctccttacct agacaatgag
600aagagcaatg gaaccattat ccatgtgaaa gggaaacacc tttgtccaag
tcccctattt 660cccggacctt ctaagccctt ttgggtgctg gtggtggttg
gtggagtcct ggcttgctat 720agcttgctag taacagtggc ctttattatt
ttctgggtga ggagtaagag gagcaggctc 780ctgcacagtg actacatgaa
catgactccc cgccgccccg ggcccacccg caagcattac 840cagccctatg
ccccaccacg cgacttcgca gcctatcgct cctgacacgg acgcctatcc
900agaagccagc cggctggcag cccccatctg ctcaatatca ctgctctgga
taggaaatga 960ccgccatctc cagccggcca cctcaggccc ctgttgggcc
accaatgcca atttttctcg 1020agtgactaga ccaaatatca agatcatttt
gagactctga aatgaagtaa aagagatttc 1080ctgtgacagg ccaagtctta
cagtgccatg gcccacattc caacttacca tgtacttagt 1140gacttgactg
agaagttagg gtagaaaaca aaaagggagt ggattctggg agcctcttcc
1200ctttctcact cacctgcaca tctcagtcaa gcaaagtgtg gtatccacag
acattttagt 1260tgcagaagaa aggctaggaa atcattcctt ttggttaaat
gggtgtttaa tcttttggtt 1320agtgggttaa acggggtaag ttagagtagg
gggagggata ggaagacata tttaaaaacc 1380attaaaacac tgtctcccac
tcatgaaatg agccacgtag ttcctattta atgctgtttt 1440cctttagttt
agaaatacat agacattgtc ttttatgaat tctgatcata tttagtcatt
1500ttgaccaaat gagggatttg gtcaaatgag ggattccctc aaagcaatat
caggtaaacc 1560aagttgcttt cctcactccc tgtcatgaga cttcagtgtt
aatgttcaca atatactttc 1620gaaagaataa aatagttctc ctacatgaag
aaagaatatg tcaggaaata aggtcacttt 1680atgtcaaaat tatttgagta
ctatgggacc tggcgcagtg gctcatgctt gtaatcccag 1740cactttggga
ggccgaggtg ggcagatcac ttgagatcag gaccagcctg gtcaagatgg
1800tgaaactccg tctgtactaa aaatacaaaa tttagcttgg cctggtggca
ggcacctgta 1860atcccagctg cccaagaggc tgaggcatga gaatcgcttg
aacctggcag gcggaggttg 1920cagtgagccg agatagtgcc acagctctcc
agcctgggcg acagagtgag actccatctc 1980aaacaacaac aacaacaaca
acaacaacaa caaaccacaa aattatttga gtactgtgaa 2040ggattatttg
tctaacagtt cattccaatc agaccaggta ggagctttcc tgtttcatat
2100gtttcagggt tgcacagttg gtctctttaa tgtcggtgtg gagatccaaa
gtgggttgtg 2160gaaagagcgt ccataggaga agtgagaata ctgtgaaaaa
gggatgttag cattcattag 2220agtatgagga tgagtcccaa gaaggttctt
tggaaggagg acgaatagaa tggagtaatg 2280aaattcttgc catgtgctga
ggagatagcc agcattaggt gacaatcttc cagaagtggt 2340caggcagaag
gtgccctggt gagagctcct ttacagggac tttatgtggt ttagggctca
2400gagctccaaa actctgggct cagctgctcc tgtaccttgg aggtccattc
acatgggaaa 2460gtattttgga atgtgtcttt tgaagagagc atcagagttc
ttaagggact gggtaaggcc 2520tgaccctgaa atgaccatgg atatttttct
acctacagtt tgagtcaact agaatatgcc 2580tggggacctt gaagaatggc
ccttcagtgg ccctcaccat ttgttcatgc ttcagttaat 2640tcaggtgttg
aaggagctta ggttttagag gcacgtagac ttggttcaag tctcgttagt
2700agttgaatag cctcaggcaa gtcactgccc acctaagatg atggttcttc
aactataaaa 2760tggagataat ggttacaaat gtctcttcct atagtataat
ctccataagg gcatggccca 2820agtctgtctt tgactctgcc tatccctgac
atttagtagc atgcccgaca tacaatgtta 2880gctattggta ttattgccat
atagataaat tatgtataaa aattaaactg ggcaatagcc 2940taagaagggg
ggaatattgt aacacaaatt taaacccact acgcagggat gaggtgctat
3000aatatgagga ccttttaact tccatcattt tcctgtttct tgaaatagtt
tatcttgtaa 3060tgaaatataa ggcacctccc acttttatgt atagaaagag
gtcttttaat ttttttttaa 3120tgtgagaagg aagggaggag taggaatctt
gagattccag atcgaaaata ctgtactttg 3180gttgattttt aagtgggctt
ccattccatg gatttaatca gtcccaagaa gatcaaactc 3240agcagtactt
gggtgctgaa gaactgttgg atttaccctg gcacgtgtgc cacttgccag
3300cttcttgggc acacagagtt cttcaatcca agttatcaga ttgtatttga
aaatgacaga 3360gctggagagt tttttgaaat ggcagtggca aataaataaa
tacttttttt taaatggaaa 3420gacttgatct atggtaataa atgattttgt
tttctgactg gaaaaatagg cctactaaag 3480atgaatcaca cttgagatgt
ttcttactca ctctgcacag aaacaaagaa gaaatgttat 3540acagggaagt
ccgttttcac tattagtatg aaccaagaaa tggttcaaaa acagtggtag
3600gagcaatgct ttcatagttt cagatatggt agttatgaag aaaacaatgt
catttgctgc 3660tattattgta agagtcttat aattaatggt actcctataa
tttttgattg tgagctcacc 3720tatttgggtt aagcatgcca atttaaagag
accaagtgta tgtacattat gttctacata 3780ttcagtgata aaattactaa
actactatat gtctgcttta aatttgtact ttaatattgt 3840cttttggtat
taagaaagat atgctttcag aatagatatg cttcgctttg gcaaggaatt
3900tggatagaac ttgctattta aaagaggtgt ggggtaaatc cttgtataaa
tctccagttt 3960agcctttttt gaaaaagcta gactttcaaa tactaatttc
acttcaagca gggtacgttt 4020ctggtttgtt tgcttgactt cagtcacaat
ttcttatcag accaatggct gacctctttg 4080agatgtcagg ctaggcttac
ctatgtgttc tgtgtcatgt gaatgctgag aagtttgaca 4140gagatccaac
ttcagccttg accccatcag tccctcgggt taactaactg agccaccggt
4200cctcatggct attttaatga gggtattgat ggttaaatgc atgtctgatc
ccttatccca 4260gccatttgca ctgccagctg ggaactatac cagacctgga
tactgatccc aaagtgttaa 4320attcaactac atgctggaga ttagagatgg
tgccaataaa ggacccagaa ccaggatctt 4380gattgctata gacttattaa
taatccaggt caaagagagt gacacacact ctctcaagac 4440ctggggtgag
ggagtctgtg ttatctgcaa ggccatttga ggctcagaaa gtctctcttt
4500cctatagata tatgcatact ttctgacata taggaatgta tcaggaatac
tcaaccatca 4560caggcatgtt cctacctcag ggcctttaca tgtcctgttt
actctgtcta gaatgtcctt 4620ctgtagatga cctggcttgc ctcgtcaccc
ttcaggtcct tgctcaagtg tcatcttctc 4680ccctagttaa actaccccac
accctgtctg ctttccttgc ttatttttct ccatagcatt 4740ttaccatctc
ttacattaga catttttctt atttatttgt agtttataag cttcatgagg
4800caagtaactt tgctttgttt cttgctgtat ctccagtgcc cagagcagtg
cctggtatat 4860aataaatatt tattgactga gtgaaaaaaa aaaaaaaaaa
490041220PRTHomo sapiens 41Met Leu Arg Leu Leu Leu Ala Leu Asn Leu
Phe Pro Ser Ile Gln Val 1 5 10 15 Thr Gly Asn Lys Ile Leu Val Lys
Gln Ser Pro Met Leu Val Ala Tyr 20 25 30 Asp Asn Ala Val Asn Leu
Ser Cys Lys Tyr Ser Tyr Asn Leu Phe Ser 35 40 45 Arg Glu Phe Arg
Ala Ser Leu His Lys Gly Leu Asp Ser Ala Val Glu 50 55 60 Val Cys
Val Val Tyr Gly Asn Tyr Ser Gln Gln Leu Gln Val Tyr Ser 65 70 75 80
Lys Thr Gly Phe Asn Cys Asp Gly Lys Leu Gly Asn Glu Ser Val Thr 85
90 95 Phe Tyr Leu Gln Asn Leu Tyr Val Asn Gln Thr Asp Ile Tyr Phe
Cys 100 105 110 Lys Ile Glu Val Met Tyr Pro Pro Pro Tyr Leu Asp Asn
Glu Lys Ser 115 120 125 Asn Gly Thr Ile Ile His Val Lys Gly Lys His
Leu Cys Pro Ser Pro 130 135 140 Leu Phe Pro Gly Pro Ser Lys Pro Phe
Trp Val Leu Val Val Val Gly 145 150 155 160 Gly Val Leu Ala Cys Tyr
Ser Leu Leu Val Thr Val Ala Phe Ile Ile 165 170 175 Phe Trp Val Arg
Ser Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met 180 185 190 Asn Met
Thr Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro 195 200 205
Tyr Ala Pro Pro Arg Asp Phe Ala Ala Tyr Arg Ser 210 215 220
421906DNAHomo sapiens 42ccaagtcaca tgattcagga ttcaggggga gaatccttct
tggaacagag atgggcccag 60aactgaatca gatgaagaga gataaggtgt gatgtgggga
agactatata aagaatggac 120ccagggctgc agcaagcact caacggaatg
gcccctcctg gagacacagc catgcatgtg 180ccggcgggct ccgtggccag
ccacctgggg accacgagcc gcagctattt ctatttgacc 240acagccactc
tggctctgtg ccttgtcttc acggtggcca ctattatggt gttggtcgtt
300cagaggacgg actccattcc caactcacct gacaacgtcc ccctcaaagg
aggaaattgc 360tcagaagacc tcttatgtat cctgaaaaga gctccattca
agaagtcatg ggcctacctc 420caagtggcaa agcatctaaa caaaaccaag
ttgtcttgga acaaagatgg cattctccat 480ggagtcagat atcaggatgg
gaatctggtg atccaattcc ctggtttgta cttcatcatt 540tgccaactgc
agtttcttgt acaatgccca aataattctg tcgatctgaa gttggagctt
600ctcatcaaca agcatatcaa aaaacaggcc ctggtgacag tgtgtgagtc
tggaatgcaa 660acgaaacacg tataccagaa tctctctcaa ttcttgctgg
attacctgca ggtcaacacc 720accatatcag tcaatgtgga tacattccag
tacatagata caagcacctt tcctcttgag 780aatgtgttgt ccatcttctt
atacagtaat tcagactgaa cagtttctct tggccttcag 840gaagaaagcg
cctctctacc atacagtatt tcatccctcc aaacacttgg gcaaaaagaa
900aactttagac caagacaaac tacacagggt attaaatagt atacttctcc
ttctgtctct 960tggaaagata cagctccagg gttaaaaaga gagtttttag
tgaagtatct ttcagatagc 1020aggcagggaa gcaatgtagt gtggtgggca
gagccccaca cagaatcaga agggatgaat 1080ggatgtccca gcccaaccac
taattcactg tatggtcttg atctatttct tctgttttga 1140gagcctccag
ttaaaatggg gcttcagtac cagagcagct agcaactctg ccctaatggg
1200aaatgaaggg gagctgggtg tgagtgttta cactgtgccc ttcacgggat
acttctttta 1260tctgcagatg gcctaatgct tagttgtcca agtcgcgatc
aaggactctc tcacacagga 1320aacttcccta tactggcaga tacacttgtg
actgaaccat gcccagttta tgcctgtctg 1380actgtcactc tggcactagg
aggctgatct tgtactccat atgaccccac ccctaggaac 1440ccccagggaa
aaccaggctc ggacagcccc ctgttcctga gatggaaagc acaaatttaa
1500tacaccacca caatggaaaa caagttcaaa gacttttact tacagatcct
ggacagaaag 1560ggcataatga gtctgaaggg cagtcctcct tctccaggtt
acatgaggca ggaataagaa 1620gtcagacaga gacagcaaga cagttaacaa
cgtaggtaaa gaaatagggt gtggtcactc 1680tcaattcact ggcaaatgcc
tgaatggtct gtctgaagga agcaacagag aagtggggaa 1740tccagtctgc
taggcaggaa agatgcctct aagttcttgt ctctggccag aggtgtggta
1800tagaaccaga aacccatatc aagggtgact aagcccggct tccggtatga
gaaattaaac 1860ttgtatacaa aatggttgcc aaggcaacat aaaattataa gaattc
190643234PRTHomo sapiens 43Met Asp Pro Gly Leu Gln Gln Ala Leu Asn
Gly Met Ala Pro Pro Gly 1 5 10 15 Asp Thr Ala Met His Val Pro Ala
Gly Ser Val Ala Ser His Leu Gly 20 25 30 Thr Thr Ser Arg Ser Tyr
Phe Tyr Leu Thr Thr Ala Thr Leu Ala Leu 35 40 45 Cys Leu Val Phe
Thr Val Ala Thr Ile Met Val Leu Val Val Gln Arg 50 55 60 Thr Asp
Ser Ile Pro Asn Ser Pro Asp Asn Val Pro Leu Lys Gly Gly 65 70 75 80
Asn Cys Ser Glu Asp Leu Leu Cys Ile Leu Lys Arg Ala Pro Phe Lys 85
90 95 Lys Ser Trp Ala Tyr Leu Gln Val Ala Lys His Leu Asn Lys Thr
Lys 100 105 110 Leu Ser Trp Asn Lys Asp Gly Ile Leu His Gly Val Arg
Tyr Gln Asp 115 120 125 Gly Asn Leu Val Ile Gln Phe Pro Gly Leu Tyr
Phe Ile Ile Cys Gln 130 135 140 Leu Gln Phe Leu Val Gln Cys Pro Asn
Asn Ser Val Asp Leu Lys Leu 145 150 155 160 Glu Leu Leu Ile Asn Lys
His Ile Lys Lys Gln Ala Leu Val Thr Val 165 170 175 Cys Glu Ser Gly
Met Gln Thr Lys His Val Tyr Gln Asn Leu Ser Gln 180 185 190 Phe Leu
Leu Asp Tyr Leu Gln Val Asn Thr Thr Ile Ser Val Asn Val 195 200 205
Asp Thr Phe Gln Tyr Ile Asp Thr Ser Thr Phe Pro Leu Glu Asn Val 210
215 220 Leu Ser Ile Phe Leu Tyr Ser Asn Ser Asp 225 230
441629DNAHomo sapiens 44tttcctgggc ggggccaagg ctggggcagg ggagtcagca
gaggcctcgc tcgggcgccc 60agtggtcctg ccgcctggtc tcacctcgct atggttcgtc
tgcctctgca gtgcgtcctc 120tggggctgct tgctgaccgc tgtccatcca
gaaccaccca ctgcatgcag agaaaaacag 180tacctaataa acagtcagtg
ctgttctttg tgccagccag gacagaaact ggtgagtgac 240tgcacagagt
tcactgaaac ggaatgcctt ccttgcggtg aaagcgaatt cctagacacc
300tggaacagag agacacactg ccaccagcac aaatactgcg accccaacct
agggcttcgg 360gtccagcaga agggcacctc agaaacagac accatctgca
cctgtgaaga aggctggcac 420tgtacgagtg aggcctgtga gagctgtgtc
ctgcaccgct catgctcgcc cggctttggg 480gtcaagcaga ttgctacagg
ggtttctgat accatctgcg agccctgccc agtcggcttc 540ttctccaatg
tgtcatctgc tttcgaaaaa tgtcaccctt ggacaagctg tgagaccaaa
600gacctggttg tgcaacaggc aggcacaaac aagactgatg ttgtctgtgg
tccccaggat 660cggctgagag ccctggtggt gatccccatc atcttcggga
tcctgtttgc catcctcttg 720gtgctggtct ttatcaaaaa ggtggccaag
aagccaacca ataaggcccc ccaccccaag 780caggaacccc aggagatcaa
ttttcccgac gatcttcctg gctccaacac tgctgctcca 840gtgcaggaga
ctttacatgg atgccaaccg gtcacccagg aggatggcaa
agagagtcgc 900atctcagtgc aggagagaca gtgaggctgc acccacccag
gagtgtggcc acgtgggcaa 960acaggcagtt ggccagagag cctggtgctg
ctgctgctgt ggcgtgaggg tgaggggctg 1020gcactgactg ggcatagctc
cccgcttctg cctgcacccc tgcagtttga gacaggagac 1080ctggcactgg
atgcagaaac agttcacctt gaagaacctc tcacttcacc ctggagccca
1140tccagtctcc caacttgtat taaagacaga ggcagaagtt tggtggtggt
ggtgttgggg 1200tatggtttag taatatccac cagaccttcc gatccagcag
tttggtgccc agagaggcat 1260catggtggct tccctgcgcc caggaagcca
tatacacaga tgcccattgc agcattgttt 1320gtgatagtga acaactggaa
gctgcttaac tgtccatcag caggagactg gctaaataaa 1380attagaatat
atttatacaa cagaatctca aaaacactgt tgagtaagga aaaaaaggca
1440tgctgctgaa tgatgggtat ggaacttttt aaaaaagtac atgcttttat
gtatgtatat 1500tgcctatgga tatatgtata aatacaatat gcatcatata
ttgatataac aagggttctg 1560gaagggtaca cagaaaaccc acagctcgaa
gagtggtgac gtctggggtg gggaagaagg 1620gtctggggg 162945277PRTHomo
sapiens 45Met Val Arg Leu Pro Leu Gln Cys Val Leu Trp Gly Cys Leu
Leu Thr 1 5 10 15 Ala Val His Pro Glu Pro Pro Thr Ala Cys Arg Glu
Lys Gln Tyr Leu 20 25 30 Ile Asn Ser Gln Cys Cys Ser Leu Cys Gln
Pro Gly Gln Lys Leu Val 35 40 45 Ser Asp Cys Thr Glu Phe Thr Glu
Thr Glu Cys Leu Pro Cys Gly Glu 50 55 60 Ser Glu Phe Leu Asp Thr
Trp Asn Arg Glu Thr His Cys His Gln His 65 70 75 80 Lys Tyr Cys Asp
Pro Asn Leu Gly Leu Arg Val Gln Gln Lys Gly Thr 85 90 95 Ser Glu
Thr Asp Thr Ile Cys Thr Cys Glu Glu Gly Trp His Cys Thr 100 105 110
Ser Glu Ala Cys Glu Ser Cys Val Leu His Arg Ser Cys Ser Pro Gly 115
120 125 Phe Gly Val Lys Gln Ile Ala Thr Gly Val Ser Asp Thr Ile Cys
Glu 130 135 140 Pro Cys Pro Val Gly Phe Phe Ser Asn Val Ser Ser Ala
Phe Glu Lys 145 150 155 160 Cys His Pro Trp Thr Ser Cys Glu Thr Lys
Asp Leu Val Val Gln Gln 165 170 175 Ala Gly Thr Asn Lys Thr Asp Val
Val Cys Gly Pro Gln Asp Arg Leu 180 185 190 Arg Ala Leu Val Val Ile
Pro Ile Ile Phe Gly Ile Leu Phe Ala Ile 195 200 205 Leu Leu Val Leu
Val Phe Ile Lys Lys Val Ala Lys Lys Pro Thr Asn 210 215 220 Lys Ala
Pro His Pro Lys Gln Glu Pro Gln Glu Ile Asn Phe Pro Asp 225 230 235
240 Asp Leu Pro Gly Ser Asn Thr Ala Ala Pro Val Gln Glu Thr Leu His
245 250 255 Gly Cys Gln Pro Val Thr Gln Glu Asp Gly Lys Glu Ser Arg
Ile Ser 260 265 270 Val Gln Glu Arg Gln 275 46913DNAHomo sapiens
46ccagagaggg gcaggctggt cccctgacag gttgaagcaa gtagacgccc aggagccccg
60ggagggggct gcagtttcct tccttccttc tcggcagcgc tccgcgcccc catcgcccct
120cctgcgctag cggaggtgat cgccgcggcg atgccggagg agggttcggg
ctgctcggtg 180cggcgcaggc cctatgggtg cgtcctgcgg gctgctttgg
tcccattggt cgcgggcttg 240gtgatctgcc tcgtggtgtg catccagcgc
ttcgcacagg ctcagcagca gctgccgctc 300gagtcacttg ggtgggacgt
agctgagctg cagctgaatc acacaggacc tcagcaggac 360cccaggctat
actggcaggg gggcccagca ctgggccgct ccttcctgca tggaccagag
420ctggacaagg ggcagctacg tatccatcgt gatggcatct acatggtaca
catccaggtg 480acgctggcca tctgctcctc cacgacggcc tccaggcacc
accccaccac cctggccgtg 540ggaatctgct ctcccgcctc ccgtagcatc
agcctgctgc gtctcagctt ccaccaaggt 600tgtaccattg cctcccagcg
cctgacgccc ctggcccgag gggacacact ctgcaccaac 660ctcactggga
cacttttgcc ttcccgaaac actgatgaga ccttctttgg agtgcagtgg
720gtgcgcccct gaccactgct gctgattagg gttttttaaa ttttatttta
ttttatttaa 780gttcaagaga aaaagtgtac acacaggggc cacccggggt
tggggtggga gtgtggtggg 840gggtagtggt ggcaggacaa gagaaggcat
tgagcttttt ctttcatttt cctattaaaa 900aatacaaaaa tca 91347193PRTHomo
sapiens 47Met Pro Glu Glu Gly Ser Gly Cys Ser Val Arg Arg Arg Pro
Tyr Gly 1 5 10 15 Cys Val Leu Arg Ala Ala Leu Val Pro Leu Val Ala
Gly Leu Val Ile 20 25 30 Cys Leu Val Val Cys Ile Gln Arg Phe Ala
Gln Ala Gln Gln Gln Leu 35 40 45 Pro Leu Glu Ser Leu Gly Trp Asp
Val Ala Glu Leu Gln Leu Asn His 50 55 60 Thr Gly Pro Gln Gln Asp
Pro Arg Leu Tyr Trp Gln Gly Gly Pro Ala 65 70 75 80 Leu Gly Arg Ser
Phe Leu His Gly Pro Glu Leu Asp Lys Gly Gln Leu 85 90 95 Arg Ile
His Arg Asp Gly Ile Tyr Met Val His Ile Gln Val Thr Leu 100 105 110
Ala Ile Cys Ser Ser Thr Thr Ala Ser Arg His His Pro Thr Thr Leu 115
120 125 Ala Val Gly Ile Cys Ser Pro Ala Ser Arg Ser Ile Ser Leu Leu
Arg 130 135 140 Leu Ser Phe His Gln Gly Cys Thr Ile Ala Ser Gln Arg
Leu Thr Pro 145 150 155 160 Leu Ala Arg Gly Asp Thr Leu Cys Thr Asn
Leu Thr Gly Thr Leu Leu 165 170 175 Pro Ser Arg Asn Thr Asp Glu Thr
Phe Phe Gly Val Gln Trp Val Arg 180 185 190 Pro 48723DNAHomo
sapiens 48atggaggaga gtgtcgtacg gccctcagtg tttgtggtgg atggacagac
cgacatccca 60ttcacgaggc tgggacgaag ccaccggaga cagtcgtgca gtgtggcccg
ggtgggtctg 120ggtctcttgc tgttgctgat gggggccggg ctggccgtcc
aaggctggtt cctcctgcag 180ctgcactggc gtctaggaga gatggtcacc
cgcctgcctg acggacctgc aggctcctgg 240gagcagctga tacaagagcg
aaggtctcac gaggtcaacc cagcagcgca tctcacaggg 300gccaactcca
gcttgaccgg cagcgggggg ccgctgttat gggagactca gctgggcctg
360gccttcctga ggggcctcag ctaccacgat ggggcccttg tggtcaccaa
agctggctac 420tactacatct actccaaggt gcagctgggc ggtgtgggct
gcccgctggg cctggccagc 480accatcaccc acggcctcta caagcgcaca
ccccgctacc ccgaggagct ggagctgttg 540gtcagccagc agtcaccctg
cggacgggcc accagcagct cccgggtctg gtgggacagc 600agcttcctgg
gtggtgtggt acacctggag gctggggagg aggtggtcgt ccgtgtgctg
660gatgaacgcc tggttcgact gcgtgatggt acccggtctt acttcggggc
tttcatggtg 720tga 72349240PRTHomo sapiens 49Met Glu Glu Ser Val Val
Arg Pro Ser Val Phe Val Val Asp Gly Gln 1 5 10 15 Thr Asp Ile Pro
Phe Thr Arg Leu Gly Arg Ser His Arg Arg Gln Ser 20 25 30 Cys Ser
Val Ala Arg Val Gly Leu Gly Leu Leu Leu Leu Leu Met Gly 35 40 45
Ala Gly Leu Ala Val Gln Gly Trp Phe Leu Leu Gln Leu His Trp Arg 50
55 60 Leu Gly Glu Met Val Thr Arg Leu Pro Asp Gly Pro Ala Gly Ser
Trp 65 70 75 80 Glu Gln Leu Ile Gln Glu Arg Arg Ser His Glu Val Asn
Pro Ala Ala 85 90 95 His Leu Thr Gly Ala Asn Ser Ser Leu Thr Gly
Ser Gly Gly Pro Leu 100 105 110 Leu Trp Glu Thr Gln Leu Gly Leu Ala
Phe Leu Arg Gly Leu Ser Tyr 115 120 125 His Asp Gly Ala Leu Val Val
Thr Lys Ala Gly Tyr Tyr Tyr Ile Tyr 130 135 140 Ser Lys Val Gln Leu
Gly Gly Val Gly Cys Pro Leu Gly Leu Ala Ser 145 150 155 160 Thr Ile
Thr His Gly Leu Tyr Lys Arg Thr Pro Arg Tyr Pro Glu Glu 165 170 175
Leu Glu Leu Leu Val Ser Gln Gln Ser Pro Cys Gly Arg Ala Thr Ser 180
185 190 Ser Ser Arg Val Trp Trp Asp Ser Ser Phe Leu Gly Gly Val Val
His 195 200 205 Leu Glu Ala Gly Glu Glu Val Val Val Arg Val Leu Asp
Glu Arg Leu 210 215 220 Val Arg Leu Arg Asp Gly Thr Arg Ser Tyr Phe
Gly Ala Phe Met Val 225 230 235 240
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